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Endocrinology Vol. 145, No. 9 4192-4203
Copyright © 2004 by The Endocrine Society

Tumor Necrosis Factor {alpha} Stimulates MUC1 Synthesis and Ectodomain Release in a Human Uterine Epithelial Cell Line

Amantha Thathiah, Melissa Brayman, Neeraja Dharmaraj, JoAnne J. Julian, Errin L. Lagow and Daniel D. Carson

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

Address all correspondence and requests for reprints to: Daniel D. Carson, Department of Biological Sciences, University of Delaware, Newark, Delaware 19716. E-mail: dcarson{at}udel.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regulation of MUC1 expression and removal is a salient feature of embryo implantation, bacterial clearance, and tumor progression. In some species, embryo implantation is accompanied by a transcriptional decline in uterine epithelial expression of MUC1. In other species, MUC1 is locally removed at blastocyst attachment sites, suggesting a proteolytic activity. Previously, we demonstrated that MUC1 is proteolytically released from the surface of a human uterine epithelial cell line, HES, and identified TNF{alpha} converting enzyme/a disintegrin and metalloprotease 17 as a constitutive and phorbol ester-stimulated MUC1 sheddase. The aims of the current study were to test the ability of soluble factors elevated during the periimplantation interval in vivo to stimulate ectodomain shedding of MUC1 from HES uterine epithelial cells and to characterize the nature of this proteolytic activity(ies). We identified TNF{alpha} as a prospective endogenous stimulus of MUC1 ectodomain release and of MUC1 and TNF{alpha} converting enzyme/a disintegrin and metalloprotease 17 expression. Moreover, we established that TNF{alpha}-stimulated MUC1 shedding occurs independently of increased de novo protein synthesis and demonstrated that the TNF{alpha}-induced increase in MUC1 gene expression is mediated through the {kappa}B site in the MUC1 promoter. Finally, we determined that the TNF{alpha}-sensitive MUC1 sheddase is inhibited by the metalloprotease inhibitor, TNF{alpha} protease inhibitor (TAPI), and the endogenous tissue inhibitor of metalloprotease-3. Collectively, these studies provide the initial in vitro characterization of a putative physiological stimulus of MUC1 ectodomain release and establish the nature of the metalloproteolytic activity(ies) involved.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN ALL MAMMALS, EMBRYO implantation ultimately involves direct interaction of the blastocyst with the luminal epithelium of the uterus (1). Thus, a fundamental need for cooperation between the blastocyst and endometrium arises, and synchronization in the development of the embryo and the uterus is essential. The embryo must reach the blastocyst stage and arrive in the uterus at the proper time for nidation to occur. Similarly, the uterus undergoes a series of hormone-dependent changes in cellular proliferation and differentiation, allowing it to become receptive to a prospective embryo. However, under most conditions, the apical surface of uterine epithelia is protected by a thick glycocalyx composed largely of mucins. MUC1, a transmembrane mucin, is an important component of the uterine glycocalyx and is characterized by an extracellular domain that consists of a series of 20-amino acid tandem repeats enriched in serine, threonine, and proline residues. These features give rise to a linear and fairly rigid structure with the potential for extensive O-linked glycosylation. The fully glycosylated MUC1 protein extends 200–500 nm above the cell surface, far beyond typical cell surface components, and is thought to prevent embryo attachment through steric hindrance (2, 3). A major challenge faced by the uterus during the receptive phase is to maintain this protective barrier while creating an environment conducive to blastocyst attachment.

In the majority of species examined, a high level of MUC1 expression correlates with a uterine state of nonreceptivity to blastocyst attachment. Accordingly, MUC1 down-regulation corresponds with the period of uterine receptivity in many species, including rodents and nonhuman primates (4, 5, 6, 7); however, in rabbits and humans, MUC1 appears to be abundantly expressed during the receptive phase (8, 9). The presence of the blastocyst in the rabbit endometrium triggers a localized reduction of MUC1 only at the site of implantation (8). A transmembrane metalloprotease, a disintegrin and metalloprotease (ADAM) 9, accumulates at the site of blastocyst attachment and MUC1 loss (10) and has been implicated in the implantation process in rabbits (11). These local responses suggest that factors secreted by or expressed on the blastocyst surface might stimulate down-regulation and/or release of MUC1 through activation of cell-surface proteases. Humans, like rabbits, maintain a high level of MUC1 expression throughout the receptive phase (9). It is unclear what strategy humans have adopted to facilitate blastocyst attachment. It has been suggested that MUC1 may actually promote embryo attachment in humans (12). However, an in vitro study of cultured human uterine epithelial cells demonstrated a local loss of MUC1 at the site of human blastocyst attachment (13). This latter finding is consistent with an induced loss of MUC1 at the site of attachment, perhaps triggered by the blastocyst itself or by a factor(s) produced by the blastocyst and mediated through activation of a uterine cell-surface protease. Consequently, it is important to determine whether a component(s) involved in maternal-embryonic dialogue (i.e. a factor(s) expressed and/or secreted by the human endometrium and/or blastocyst) triggers a reduction of MUC1 expression in uterine epithelia, creating a favorable environment to blastocyst attachment and the establishment of pregnancy. In this regard, several putative attachment molecules have been identified as a result of their expression in the uterine luminal epithelium and/or the embryonic trophectoderm during the receptive phase, including numerous cytokines and growth factors such as leukemia inhibitory factor (LIF), IL-1, IL-11, and heparin-binding epidermal growth factor, the hormones calcitonin and human chorionic gonadotropin, and the cyclooxygenase (COX)-2-derived prostaglandins (PGs) (reviewed in Refs.14 and 15). Through binding to specific receptors, these factors may activate molecular changes in the expression pattern of adhesion and antiadhesion molecules essential for attachment of the blastocyst to the uterine epithelium.

TNF{alpha} is a proinflammatory cytokine proposed to play a functional role in implantation. In humans, TNF{alpha} is expressed in and secreted by the receptive endometrium (16, 17, 18, 19) and has been detected in the conditioned medium of human preimplantation embryos and blastocysts (16, 20). Human preimplantation stage embryos (21) and endometrial epithelial cells (19) also express TNF receptors (TNFR) I and II. Interestingly, TNF{alpha} stimulates expression of several proteases, including the membrane-type matrix metalloprotease (MT-MMP), MT1-MMP (22), matrix metalloprotease (MMP)-9 (23), and urokinase-type plasminogen activator (24), a protease involved in the activation of plasmin and implicated in the activation of MMPs (25). The latter finding was observed in cultured human cytotrophoblasts, suggesting a potential regulatory role for this cytokine in trophoblast attachment to and invasion through the uterine epithelium. Mice deficient in TNF{alpha} and in both the TNFRI and TNFRII do not display reduced reproductive capacity (26); however, the methods of MUC1 removal during implantation differ between mice and humans.

The purpose of the current study was to test the ability of physiologically relevant soluble factors that are elevated during the periimplantation period in vivo to induce ectodomain release of MUC1 from the HES uterine epithelial cell line, an in vitro model of human uterine epithelia, and to characterize the nature of these proteolytic activities. In this study, we identify TNF{alpha} as a prospective endogenous stimulus of MUC1 ectodomain release and synthesis in a human uterine epithelial cell line, HES, and establish that TNF{alpha}-mediated MUC1 shedding occurs independently of increased de novo protein synthesis and is at least partially mediated through activation of protein kinase C (PKC). Furthermore, we demonstrate that the TNF{alpha}-stimulated increase in MUC1 protein synthesis is mediated through the {kappa}B site in the MUC1 promoter and determine that the TNF{alpha}-sensitive MUC1 sheddase is inhibited by the synthetic hydroxamate-based metalloprotease inhibitor, TNF{alpha} protease inhibitor (TAPI), and the endogenous tissue inhibitor of metalloprotease (TIMP)-3, but is unaffected by TIMP-1 or various serine, cysteine, and aspartyl protease inhibitors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Phorbol-12 myristate 13-acetate (PMA), leupeptin, pepstatin A, E-64, and protein G-Sepharose were obtained from Sigma Chemical Co. (St. Louis, MO). The PKC inhibitor, calphostin C, brefeldin A, and monensin were purchased from Calbiochem (San Diego, CA). GM6001, rabbit anti-TNF{alpha} converting enzyme (TACE) antibody, TIMP-1, and TIMP-2 were obtained from Chemicon (Temecula, CA). TIMP-3 was purchased from R&D Systems (Minneapolis, MN). Human TNF{alpha} was obtained from Roche Molecular Biochemicals (Nutley, NJ). Affinity-purified mouse IgG and rabbit IgG were obtained from Zymed (San Francisco, CA). A mouse monoclonal antibody specific for a tandem repeat epitope in the extracellular domain of MUC1, 214D4, was kindly provided by Dr. John Hilkens (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The metalloprotease inhibitor, TAPI, was kindly provided by Dr. Roy Black and Dr. John Doedens (Amgen, Seattle, WA).

Cell culture 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 DMEM (Life Technologies, Carlsbad, CA) supplemented with 10% (vol/vol) charcoal-stripped fetal bovine serum (Hyclone, Logan, UT), 100 µM sodium pyruvate (Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). Cells were seeded on Matrigel-coated (BD Biosciences, San Jose, CA) 24-well tissue culture plates (Costar, Acton, MA) and maintained as described until cells reached 90% confluence. The cells then were serum-starved for 24 h before the beginning of treatment. At the time of treatment, culture medium was replaced with fresh serum-free medium in the presence or the absence of TNF{alpha} (10, 25, or 100 ng/ml) and one of the following protease inhibitors: leupeptin (10 µM), pepstatin A (10 µM), E-64 (10 µM), TAPI (100 µM), GM6001 (50 µM), TIMP-1 (20 µg/ml), TIMP-2 (20 µg/ml), TIMP-3 (20 µg/ml), or the appropriate vehicle control. In other experiments, calphostin C (1 µM) was added in the presence or absence of TNF{alpha} (100 ng/ml). After 3- or 24-h incubations, the cells were examined by phase microscopy for survival and morphology, and cell lysates and culture supernatants were collected for Western blot analysis. In all cases, cell viability exceeded 95% by the Trypan blue exclusion assay.

Sample preparation, SDS-PAGE, and detection of MUC1 protein
Sample preparation, protein determination, SDS-PAGE, and Western blot analysis of MUC1 protein were performed as previously described (27). Statistical analyses were performed using one-way ANOVA and the Tukey-Kramer multiple comparisons test (GraphPad InStat program; GraphPad Software Inc., San Diego, CA).

Transient transfections and reporter assays
The 1.4MUC and 1.4mut{kappa}B plasmids were generated as previously described (28). Cells were seeded on growth factor-reduced Matrigel-coated six-well tissue culture plates and maintained as described until cells reached 60–75% confluence. Cells were serum-starved for 24 h before transient transfection. Transient transfections were performed using LipofectAMINE reagent (Life Technologies) according to the manufacturer’s instructions. Two micrograms of either the 1.4MUC or the 1.4mut{kappa}B plasmid and 0.25 µg of pRL-TK plasmid were used per well. After transfection, cells were given fresh medium containing 1% (vol/vol) charcoal-stripped fetal bovine serum and either 25 ng/ml recombinant human TNF{alpha} or vehicle (0.1% BSA in 1x PBS) for 12 h. Luciferase assays were performed using the Dual-Luciferase Assay Kit (Promega, Madison, WI) according to the manufacturer’s instructions and analyzed using a Dynex MLX Microplate Luminometer (Dynex Technologies, Gaithersburg, MD). Reporter activity was expressed as the ratio of firefly luciferase activity to Renilla luciferase activity. Statistical analyses were performed by GraphPad InStat software (GraphPad), using one-way ANOVA and the Tukey-Kramer multiple comparisons test.

RNA isolation and real-time RT-PCR
HES cells were maintained as described in six-well Matrigel-coated plates until cells reached 60–75% confluence. The cells were then serum-starved for 24 h before the beginning of treatment. At the time of treatment, culture medium was replaced with fresh serum-free medium in the presence of TNF{alpha} (25 ng/ml) or the appropriate vehicle control. Total RNA was extracted from HES cultures using the RNeasy kit from Qiagen (Valencia, CA) according to the manufacturer’s instructions. Total RNA was reverse transcribed using the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed in a final reaction volume of 20 µl, using random hexamers, for 10 min at room temperature, 15 min at 42 C, 5 min at 99 C, and 5 min at 5 C. Real-time PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen). The primer sequences used were: MUC1/Rep F-5'-gtgccccctagcagtaccg and R-5'-gacgtgcccctacaagttgg (100 bp) (29), and glyceraldehyde-3-phosphate dehydrogenase F-5'-gctgagtatgtcgtggagtc and R-5'-ttggtggtgcaggatgcatt (191 bp). A standard for MUC1/Rep was generated by cloning the PCR product into the pCR2.1 vector using the TOPO T/A cloning kit (Invitrogen, Carlsbad, CA). Isolated plasmid was linearized using EcoRV, quantitated, and diluted for use as a standard in real-time PCR, which was performed in the iCycler iQ real-time PCR detection system from Bio-Rad Laboratories, Inc. (Hercules, CA). After 13 min and 30 sec of incubation at 95 C, the cycling conditions were as follows: denature at 95 C for 1 min, anneal at 62 C (MUC1) or 58 C (glyceraldehyde-3-phosphate dehydrogenase) for 1 min, and extension for 1 min at 72 C for 40 cycles.

Brefeldin A and monensin treatment and human IL-6 ELISA
The HES cells were maintained as described earlier until the time of treatment. At the time of treatment, culture medium was replaced with fresh serum-free medium in the presence or the absence of brefeldin A (5 or 10 µg/ml) or monensin (5, 10, or 25 µg/ml) or the appropriate vehicle control. After a 24-h incubation, the cells were examined by phase microscopy for survival and morphology, and culture supernatants were collected for the ELISA. Secreted IL-6 was determined using the Quantikine human IL-6 ELISA (R&D Systems) according to the manufacturer’s directions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF{alpha} stimulates MUC1 expression and ectodomain release
MUC1 shedding was studied in a uterine epithelial cell line, HES, which abundantly expresses and readily sheds MUC1. To evaluate constitutive and induced MUC1 shedding activities, we examined the effects of various growth factors, cytokines, and hormones elevated during the periimplantation period to determine whether a potential physiological agonist could enhance release of MUC1 ectodomains from the uterine epithelial cell line, HES (Table 1Go; and data not shown). Time-course and dose-dependence studies revealed that TNF{alpha}, a proinflammatory cytokine expressed in and secreted by the receptive endometrium (17, 30, 31, 32) and found in the conditioned medium of human preimplantation blastocysts (16), significantly accelerated MUC1 release 3- to 5-fold in a time- and dose-dependent manner (Fig. 1Go, A and B). To assess whether TNF{alpha}-accelerated MUC1 release was the result of an increase in MUC1 protein synthesis, we determined the relative levels of cell-associated MUC1 after TNF{alpha} treatment by Western blot analysis. Figure 1Go, C and D, indicate that TNF{alpha} stimulates a 2- to 3-fold increase in cell-associated MUC1 and a 5- to 6-fold increase in MUC1 mRNA expression after a 24-h treatment (Fig. 1EGo), which is an effect on cell-associated MUC1 not observed after a 3-h TNF{alpha} treatment and independent of the TNF{alpha} concentration used.


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  		TABLE 1. Effect of soluble factors elevated during the periimplantation interval on MUC1 proteolytic release from the HES human uterine epithelial cell line

 


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  		FIG. 1. TNF{alpha} stimulates ectodomain release and synthesis of MUC1 in HES uterine epithelial cells. A, Western blot analysis of MUC1 expression in culture supernatants from HES cells treated with the indicated doses of TNF{alpha} or with vehicle (0.01%, wt/vol, BSA in PBS) for the specified times. A monoclonal antibody directed against a tandem repeat epitope in the ectodomain of MUC1, 214D4, was used to detect MUC1. As a result of differential glycosylation and allelic polymorphism, 214D4 recognizes at least two forms of MUC1. The migration position of myosin (205 kDa) is indicated to the right. The appearance of bands above 205 kDa corresponds to MUC1. The band that was detected at less than 205 kDa is due to nonspecific recognition by the secondary antibody. B, Shed MUC1 was quantified by densitometric analysis using an {alpha} Imager 1D Multi program (Alpha Innotech, San Leandro, CA) and is shown as a percentage of MUC1 released by cells treated with vehicle alone. Results represent the average ± SD of three independent samples. *, P < 0.05 relative to vehicle control. ***, P < 0.001 relative to vehicle control or after a 3-h TNF{alpha} treatment (100 ng/ml). C, Western blot analysis of MUC1 expression in cell lysates from HES cells treated with the indicated doses of TNF{alpha} or with vehicle (0.01%, wt/vol, BSA in PBS). D, Cell-associated MUC1 protein was quantified by densitometric analysis and is shown as a percentage of MUC1 expression in cells treated with vehicle alone. Results represent the average ± SD of three independent samples. **, P < 0.01; *, P < 0.05 relative to vehicle control. E, MUC1 mRNA expression in HES cells treated with TNF{alpha} or with vehicle (0.01%, wt/vol, BSA in PBS) for 24 h as described in Materials and Methods. RNA was extracted and real-time RT-PCR was performed using MUC1-specific primers. The experiment was performed in triplicate, and expression of MUC1 mRNA was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA content. ***, P < 0.001 relative to vehicle control.

 
TNF{alpha}-stimulated MUC1 promoter activity is mediated through the {kappa}B site
Previous MUC1 promoter studies conducted in our laboratory have demonstrated that stimulation of MUC1 expression by TNF{alpha} in breast cancer cells is mediated by the binding of nuclear factor {kappa}B p65 to the {kappa}B site in the MUC1 promoter (28). To test for similar regulation of MUC1 in a model of human endometrium, HES cells were transiently transfected with luciferase reporter constructs consisting of a segment of the 5' flanking sequence of the human MUC1 gene from –1406 to +33 or the 1.4-kb intact MUC1 promoter construct containing a specific mutation of the {kappa}B site at –589/–580. As shown in Fig. 2Go, TNF{alpha} stimulated a 3- to 4-fold increase in MUC1 promoter activity in cells transiently transfected with the intact 1.4-kb MUC1 promoter relative to vehicle-treated cells; however, specific mutation of the {kappa}B site significantly decreased TNF{alpha}-stimulated MUC1 promoter activity, indicating that TNF{alpha}-stimulated MUC1 transcriptional activity in HES cells requires the {kappa}B site in the MUC1 promoter. Mutation of the {kappa}B site also reduced basal MUC1 promoter activity. In this regard, we have determined that HES cells endogenously express TNF{alpha} that is likely to drive basal MUC1 expression in an autocrine fashion (data not shown). Nonetheless, endogenous TNF{alpha} can only account for a partial stimulation of MUC1 expression because addition of TNF{alpha} to the culture medium strongly stimulates MUC1 expression.



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  		FIG. 2. Response of the MUC1 promoter to TNF{alpha} requires the {kappa}B site at –589/–590 in HES uterine epithelial cells. HES cells were transiently transfected with the full-length MUC1 promoter construct, 1.4MUC, or the {kappa}B mutant construct and then subsequently treated with TNF{alpha} (25 ng/ml) for 24 h. Results represent the average ± SD of three independent samples. **, P < 0.01 relative to vehicle control; ***, P < 0.001 relative to TNF{alpha} treatment after transfection with the 1.4MUC construct. NF, Nuclear factor.

 
TNF{alpha} stimulates MUC1 shedding independently of increased MUC1 delivery to the cell surface
To establish whether de novo protein synthesis is required for TNF{alpha}-stimulated release of MUC1, the HES cells were treated with cycloheximide or emetine, two potent inhibitors of protein synthesis; however, time-course and dose-dependence studies indicated that effective doses of both agents were toxic to the HES cells (data not shown). As an alternate approach to determine whether TNF{alpha} enhances MUC1 shedding independently of increased MUC1 delivery to the cell surface, the HES cells were treated with the secretory pathway inhibitors, brefeldin A, which blocks vesicle budding in the endoplasmic reticulum (33), or monensin, which is thought to prevent transport beyond the medial-Golgi apparatus (34). The optimal inhibitory concentration of each agent was determined by monitoring secretion of IL-6 from HES cells (Fig. 3AGo). In both cases, IL-6 secretion was inhibited ≥ 90%. HES cells were preincubated with either agent and then treated with TNF{alpha} in the presence of brefeldin A or monensin. Observation of MUC1 in HES culture supernatants in the presence of these secretory pathway blockers indicates that TNF{alpha} stimulates ectodomain release of MUC1 independently of enhanced rates of delivery of newly synthesized MUC1 to the cell surface (Fig. 3BGo).



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  		FIG. 3. TNF{alpha}-stimulated MUC1 release is not dependent on de novo MUC1 delivery to the cell surface. A, IL-6 secretion by HES cells treated with brefeldin A (BFA; 5 µg/ml), monensin (10 µg/ml), or vehicle as described in Materials and Methods. Quantification of IL-6 secreted into the medium was determined by ELISA analysis following the manufacturer’s suggestions. Results represent the mean ± SD of four independent samples and two independent experiments. ***, P < 0.001 relative to vehicle control. B, MUC1 recovered from culture supernatants of HES cells treated with brefeldin A (BFA; 5 µg/ml) or monensin (10 µg/ml) in the presence or absence of TNF{alpha} (100 ng/ml) or vehicle (0.01% BSA in PBS). Quantification of MUC1 shed into the medium was determined by densitometric analysis and is shown as a percentage of MUC1 released by cells treated with vehicle alone. Results represent the mean ± SD of three independent samples. ***, P < 0.001; **, P < 0.01; *, P < 0.05 relative to vehicle control for each treatment group. +, P < 0.05; ++, P < 0.01 relative to TNF{alpha} (NT).

 
Shed MUC1 is devoid of a cytoplasmic tail
To confirm that the soluble MUC1 observed in the supernatants of HES cells contained ectodomains only, cell lysates and conditioned medium from TNF{alpha}-stimulated and unstimulated cells were immunoprecipitated with a polyclonal antibody, CT-1, which is specific for the cytoplasmic domain of MUC1, or with a monoclonal antibody, 214D4, which is specific for the extracellular domain of MUC1, and then they were examined by Western blot analysis, probing the membrane with 214D4 (Fig. 4Go). In agreement with other studies in various cell lines (27, 35, 36, 37), shed MUC1 was immunoprecipitated with 214D4 but not with CT-1, indicating that MUC1 in the supernatants of unstimulated and TNF{alpha}-stimulated HES cells lacks the cytoplasmic tail. Previous work has demonstrated that MUC1 constitutively released from HES is not associated with particulate elements, i.e. membrane blebs (35). Thus, the presence of ectodomain fragments in the culture supernatants from untreated cells indicates that MUC1 is released in the absence of a stimulus, and enhanced detection of MUC1 after TNF{alpha} treatment is due to increased release, i.e. shedding, rather than membrane blebbing.



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  		FIG. 4. MUC1 released from the surface of HES uterine epithelial cells is devoid of a cytoplasmic tail. HES cells were treated with TNF{alpha} (100 ng/ml) or vehicle (0.01%, wt/vol, BSA in PBS) for 24 h as described in Materials and Methods. Then MUC1 was immunoprecipitated from cell lysates (L) and culture supernatants (S) with the polyclonal antibody, CT-1, which recognizes an epitope in the cytoplasmic tail of MUC1, with 214D4, or with nonimmune mouse and rabbit control IgG. Western blot analysis of the immunoprecipitates with 214D4 indicates that the soluble MUC1 ectodomain can be immunoprecipitated with 214D4 but not with CT-1, whereas cell-associated MUC1 can be immunoprecipitated with both antibodies. This experiment was repeated twice with similar results.

 
TNF{alpha}-simulated MUC1 release is partially mediated through a PKC-dependent pathway
Phorbol ester activation of PKC has been shown to enhance ectodomain release of a diverse group of cell-surface proteins (38, 39, 40, 41, 42). Recently, the PKC activator, PMA, has been demonstrated to activate the MAPK cascade, resulting in the downstream phosphorylation of the MAPK ERK1 and 2, phosphorylation of the cytoplasmic tail of TACE/ADAM 17, and subsequent enhanced proteolytic activity (43). Previously, we demonstrated that PMA rapidly accelerated shedding of MUC1 from HES cells and identified TACE/ADAM 17 as a MUC1 sheddase (27). To initially determine whether TNF{alpha}-stimulated MUC1 shedding involves PKC activity, we examined the ability of the PKC inhibitor, calphostin C, to inhibit TNF{alpha}-enhanced MUC1 ectodomain release (Fig. 5Go). Calphostin C had no significant effect on constitutive MUC1 shedding, but it partially inhibited (30%) TNF{alpha}-stimulated MUC1 shedding, suggesting that TNF{alpha}-mediated MUC1 shedding partially involves a PKC-dependent signaling pathway.



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  		FIG. 5. TNF{alpha}-stimulated MUC1 release is partially inhibited by the PKC inhibitor, calphostin C. A, Western blot analysis of MUC1 recovered from culture supernatants of HES cells treated with TNF{alpha} (100 ng/ml) or vehicle (0.01% BSA in PBS) or calphostin C (1 µM) for 24 h as described in Materials and Methods. B, Quantification of MUC1 shed into the medium was determined by densitometric analysis and is shown as a percentage of MUC1 released by cells treated with vehicle alone. Results represent the mean ± SD of three independent samples. ***, P < 0.001 relative to vehicle control; **, P < 0.01 relative to TNF{alpha} stimulation.

 
A metalloprotease mediates TNF{alpha}-stimulated MUC1 release
To characterize the activity mediating TNF{alpha}-stimulated MUC1 cell surface release, a series of protease inhibitors were examined for their ability to modulate MUC1 shedding (Table 2Go). Inhibitors of serine (leupeptin), cysteine (E-64), and aspartyl (pepstatin A) proteases had no effect on constitutive or TNF{alpha}-stimulated MUC1 release. The broad-spectrum, hydroxamate-based metalloprotease inhibitor, GM6001 (Illomastat; Chemicon), also failed to inhibit basal and TNF{alpha}-stimulated MUC1 release (Fig. 6Go, A and B). In contrast, the structurally distinct hydroxamate-based metalloprotease inhibitor, TAPI, drastically diminished constitutive release of MUC1 by 78% and TNF{alpha}-stimulated release by 90% (Fig. 6Go, C and D). This synthetic peptide hydroxamate initially was designed to inhibit pro-TNF{alpha} shedding (44) but closely resembles a MMP inhibitor (45) and effectively inhibits both MMPs and ADAMs (46).


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  		TABLE 2. Protease inhibitor insensitivity of constitutive and TNF{alpha}-stimulated MUC1 shedding

 


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  		FIG. 6. TNF{alpha}-induced MUC1 release is differentially sensitive to the synthetic broad-spectrum metalloprotease inhibitors GM6001 and TAPI. A, Western blot analysis of MUC1 recovered from culture supernatants of HES cells treated with TNF{alpha} (100 ng/ml) or vehicle (0.01%, wt/vol, BSA in PBS) in the presence or absence of GM6001 (50 µM) for 24 h at 37 C. B, Quantification of MUC1 shed into the medium was determined by densitometric analysis and is shown as a percentage of MUC1 released by cells treated with vehicle alone. Results represent the mean ± SD of three independent samples. ***, P < 0.001 relative to vehicle control; ns, not significant relative to TNF{alpha} stimulation alone. C, Western blot analysis of MUC1 recovered from culture supernatants of HES cells treated with TNF{alpha} (100 ng/ml) or vehicle (0.01%, wt/vol, BSA in PBS) in the presence or absence of TAPI (100 µM) for 24 h at 37 C. D, Quantification of MUC1 shed into the medium was determined by densitometric analysis and is shown as a percentage of MUC1 released by cells treated with vehicle alone. Results represent the mean ± SD of three independent samples. ***, P < 0.001 relative to vehicle control or TNF{alpha} stimulation alone. Experiments were performed in triplicate and repeated at least twice with similar results.

 
To further characterize the MUC1 sheddase(s), we examined the ability of endogenous metalloprotease inhibitors TIMP-1, -2, and -3 (reviewed in Refs.47 and 48) to inhibit MUC1 shedding. TIMP-1 failed to inhibit constitutive MUC1 release but modestly inhibited TNF{alpha}-stimulated release (≤30%) from HES cells (Fig. 7Go, A and B). Surprisingly, TIMP-2 appeared to stimulate both constitutive and TNF{alpha}-accelerated MUC1 shedding (Fig. 7Go, C and D), and TIMP-3 significantly inhibited TNF{alpha}-enhanced shedding of MUC1 (Fig. 7Go, E and F). Thus, MUC1 shedding was inhibited by physiological antagonists of MMPs and ADAMs.



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  		FIG. 7. TNF{alpha} stimulation of MUC1 release is differentially sensitive to the endogenous inhibitors of MMPs, TIMP-1, -2, and -3. A, Western blot analysis of MUC1 recovered from culture supernatants of HES cells treated with TNF{alpha} (100 ng/ml) or vehicle (0.01%, wt/vol, BSA in PBS) in the presence or absence of TIMP-1 (20 µg/ml) for 24 h at 37 C. B, Quantification of MUC1 shed into the medium was determined by densitometric analysis and is shown as a percentage of MUC1 released by cells treated with vehicle alone. Results represent the mean ± SD of three independent samples. **, P < 0.01; ***, P < 0.001 relative to vehicle control; ns, not significant relative to TNF{alpha} stimulation alone. C, Western blot analysis of MUC1 recovered from culture supernatants of HES cells treated with TNF{alpha} (100 ng/ml) or vehicle (0.01%, wt/vol, BSA in PBS) in the presence or absence of TIMP-2 (20 µg/ml) for 24 h at 37 C. D, Quantification of MUC1 shed into the medium was determined by densitometric analysis and is shown as a percentage of MUC1 released by cells treated with vehicle alone. *, P < 0.05; ***, P < 0.001 relative to vehicle control or TNF{alpha} stimulation alone. E, Western blot analysis of MUC1 recovered from culture supernatants of HES cells treated with TNF{alpha} (100 ng/ml) or vehicle (0.01%, wt/vol, BSA in PBS) in the presence or absence of TIMP-3 (20 µg/ml) for 24 h at 37 C. F, Quantification of MUC1 shed into the medium was determined by densitometric analysis and is shown as a percentage of MUC1 released by cells treated with vehicle alone. *, P < 0.05 relative to vehicle control or TNF{alpha} stimulation alone. Experiments were performed in triplicate and repeated at least twice with similar results.

 
These results implicated members of the MT-MMP and/or ADAM family of metalloproteases as TNF{alpha}-stimulated candidate MUC1 sheddases. Examination of shedding events in cells derived from mice genetically deficient for specific ADAM proteases has permitted the identification of potential protease-mediated shedding events (42, 49, 50, 51). Using embryonic fibroblasts derived from wild-type and TACE-deficient mice (50), we previously demonstrated that, in contrast to wild-type EC-4 cells, TACE-deficient EC-2 cells do not shed MUC1 constitutively or in response to the phorbol ester PMA, implicating TACE as a constitutive and PMA-inducible MUC1 sheddase (27). To determine whether TACE/ADAM 17 is also a TNF{alpha}-stimulated MUC1 sheddase, MUC1 shedding was examined after electroporation of these cells with MUC1 cDNA or empty vector. However, transfected wild-type EC-4 and TACE-deficient EC-2 cells behaved similarly and failed to shed MUC1 after TNF{alpha} stimulation, indicating that TACE/ADAM 17 is not required for TNF{alpha}-stimulated MUC1 ectodomain release in this cellular context (data not shown).

Next we considered that TNF{alpha} might alter TACE/ADAM 17 expression as a means of regulating sheddase activity in HES cells. To examine this point, Western blot analysis was used to detect potential changes in TACE/ADAM 17 protein expression. As shown in Fig. 8Go, A and B, the mature and precursor forms of TACE/ADAM 17 were stimulated approximately 1.7- and 4-fold, respectively, after 24 h of TNF{alpha} treatment. In contrast, levels of another membrane-associated sheddase, MT1-MMP, were not affected by TNF{alpha} treatment (Fig. 8CGo). Collectively, these observations were consistent with ADAMs-type metalloproteases as mediators of TNF{alpha}-stimulated MUC1 shedding and implicated TACE/ADAM 17 as a principal MUC1 sheddase in this context.



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  		FIG. 8. TNF{alpha} enhances expression of the metalloprotease, TACE/ADAM 17, but not MT1-MMP, in HES uterine epithelial cells. A, Western blot analysis of TACE expression in cell lysates from HES cells treated with TNF{alpha} (100 ng/ml) or with vehicle (0.01%, wt/vol, BSA in PBS). An anti-TACE affinity-purified polyclonal antibody was used to detect TACE. A Jurkat cell lysate was used as a positive control for the anti-TACE antibody. B, Quantification of the immature and mature forms of TACE expression was determined by densitometric analysis and is shown relative to cells treated with vehicle alone. Results represent the mean ± SD of six independent samples and two separate experiments. ***, P < 0.001 relative to vehicle control; **, P < 0.01 relative to TNF{alpha}-stimulated precursor form of TACE. C, Western blot analysis of MT1-MMP expression in cell lysates from HES cells treated with TNF{alpha} (100 ng/ml) or with vehicle (0.01%, wt/vol, BSA in PBS). An anti-MT1-MMP affinity-purified polyclonal antibody was used to detect MT1-MMP. An HT-1080 cell lysate was used as a positive control for the anti-MT1-MMP antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Preparation of the endometrium for implantation involves a dramatic increase in endometrial mass which is regulated by the steroid hormones, estradiol and progesterone (P4). Although many of these changes may be directly initiated by these hormones binding to their receptors, there is evidence to suggest that locally produced growth factors, cytokines, and lipid mediators, acting in an autocrine, juxtacrine, or paracrine manner, mediate functions of estradiol and P4 in the preimplantation uterus in preparation for embryo attachment (reviewed in Ref.26). A study conducted with autologous human uterine epithelial cells prepared from uterine biopsies obtained during the proliferative and secretory phases of the menstrual cycle demonstrated that IL-1 induces a dose-dependent increase in TNF{alpha} production in cells prepared from the proliferative and secretory endometrium (52). In contrast, P4 stimulated TNF{alpha} production in cells prepared from the proliferative endometrium but induced a decrease in TNF{alpha} production in cells prepared from the secretory endometrium (52). Furthermore, IL-1 and TNF{alpha} additively stimulate LIF production in first trimester human decidual cells, an effect that is prevented by PKC inhibition (53).

The aim of the current study was to explore the hypothesis that potential physiological agonists of uterine and/or embryonic origin that are elevated during the periimplantation interval stimulate MUC1 release from the surface of a human uterine epithelial cell line. Heparin-binding epidermal growth factor, TNF{alpha}, LIF, IL-1ß, human chorionic gonadotropin, calcitonin, PGF2{alpha}, PGE2, and PGI2 were evaluated for their ability to stimulate MUC1 ectodomain release. Time-course and dose-dependence experiments revealed that, of the factors tested, only TNF{alpha} accelerated MUC1 shedding, implying that induced shedding of MUC1 is selective in this cell line. Interestingly, human blastocyst-conditioned medium also failed to stimulate shedding of MUC1 from the HES cells, suggesting that stable, embryo-derived soluble factors are unable to stimulate MUC1 ectodomain release (Thathiah, A., M. Meseguer, C. Simon, and D. D. Carson, unpublished observations). Alternatively, an embryonic stimulus with a limited active lifespan and/or too diluted by the in vitro culture medium may have precluded detection of an effect on MUC1 release.

TNF{alpha} not only markedly stimulates MUC1 shedding from HES cells but also stimulates an increase in MUC1 mRNA and protein expression. At the transcriptional level, this regulation may require the binding of nuclear factor {kappa}B family members to the {kappa}B site in the MUC1 promoter because mutation of this site almost completely abolished TNF{alpha}-mediated stimulation of MUC1 promoter activity. This observation is consistent with previous studies demonstrating that this site is active in cytokine-stimulated MUC1 expression in cultured normal mammary epithelium and in breast cancer cells (29). These observations suggest that TNF{alpha} concomitantly reinforces the uterine epithelial barrier to infection by stimulating MUC1 synthesis while facilitating embryo attachment by stimulating MUC1 ectodomain release from uterine epithelia in a temporally and, perhaps, spatially restricted manner. TNF{alpha} also enhances expression of TACE/ADAM 17 in HES cells, indicating that one aspect of TNF{alpha} action, in this regard, is to promote sheddase expression. In contrast, another membrane-anchored metalloprotease, MT1-MMP, was not affected by TNF{alpha} treatment.

The MUC1 ectodomain is shed from cultured cells at a basal rate and is markedly enhanced by direct PKC activation by the phorbol ester, PMA (27). We now demonstrate that specific ligand interactions are likely to involve receptor activation, given that the PKC inhibitor, calphostin C, partially inhibits TNF{alpha}-stimulated shedding of MUC1. Because this inhibition is incomplete, multiple intracellular pathways may be involved in this regulated shedding process. These results support a role for PKC activation in accelerated MUC1 shedding. Activation of either TNFRI or TNFRII can induce a phosphorylation/dephosphorylation cascade, activate phospholipase C{gamma}, alter intracellular calcium concentrations, enhance diacylglycerol production, and thus, stimulate PKC.

Serine, cysteine, and aspartate protease inhibitors had no effect on MUC1 ectodomain release; however, constitutive and TNF{alpha}-stimulated MUC1 shedding was sensitive to the hydroxamate-based metalloprotease inhibitor, TAPI, indicating the involvement of a metalloprotease(s) in constitutive and TNF{alpha}-stimulated MUC1 shedding. The endogenous MMP inhibitors, TIMPs, inhibit the known MMPs to a varying extent (reviewed in Ref.54), and several MT-MMPs are effectively inhibited by TIMP-2 and TIMP-3 (55). The TIMP inhibition profile of the MUC1 sheddase(s) suggests that it is not a known MMP or MT-MMP because neither constitutive nor stimulated MUC1 release is inhibited by TIMP-1 or TIMP-2. TIMP-3, however, is highly expressed at the maternal-fetal interface during human implantation and has been suggested to play a regulatory role in trophoblast invasion (56, 57). TIMP-3 also inhibits the metalloproteolytic-dependent shedding of L-selectin (58), syndecan-1 and -4 (59), and the IL-6 receptor (60). Moreover, TIMP-3 is the only TIMP found to bind heparan sulfate proteoglycans expressed on the cell surface (61), which may permit colocalization and interaction with cell-surface metalloproteases. Thus, the sensitivity of stimulated MUC1 release to the endogenous metalloprotease inhibitor TIMP-3 and the synthetic metalloprotease inhibitor TAPI, along with the marked stimulation of MUC1 shedding in response to TNF{alpha}, are consistent with the involvement of ADAM-type proteases, such as TACE/ADAM 17, in proteolytic release of MUC1.

In conclusion, the current studies provide the initial characterization of a potential physiological agonist of MUC1 ectodomain shedding in a model of the human uterine epithelium. These studies were conducted using an in vitro cell culture system, and therefore, the relevance of these findings with regard to human implantation in vivo are still unknown and remain to be determined. Nonetheless, we demonstrated that MUC1 shedding is stimulated in vitro from the HES uterine epithelial cell line after treatment with TNF{alpha} but not by a variety of other growth factors, cytokines, hormones, or lipid mediators that are elevated during the periimplantation interval. Additionally, we established that TNF{alpha} enhances MUC1 mRNA and protein expression and that transcriptional regulation of the TNF{alpha}-mediated response involves the {kappa}B site in the MUC1 promoter. Based on the protease inhibition profile of the sheddase(s), the TNF{alpha}-stimulated activity appears to be mediated by an ADAM-type metalloprotease, probably TACE/ADAM 17, a sheddase previously shown to mediate MUC1 shedding (28). Finally, identification of constitutive and regulated mechanisms of MUC1 shedding is another level of control in addition to transcription, translation, alternative splicing, and tissue/cell specificity. Considering the observed correlation between overexpression of MUC1, the metastatic potential of primary tumors, and poor patient survival, and the involvement of MUC1 in protection of mucosal epithelia and embryo implantation, the identification of potential physiologically relevant modulators of MUC1 synthesis and shedding should provide new opportunities for control of MUC1 expression and removal under normal and aberrant conditions.


   Acknowledgments
 
We thank Dr. John Hilkens for his generous gift of the MUC1 antibody, 214D4. We are especially grateful to Sharron Kingston and Margie Barrett for their expert secretarial and graphics assistance. We are greatly indebted to Dr. Carl P. Blobel and to members of the Carson and Farach-Carson Laboratories for many helpful discussions.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD 29963 (to D.D.C.) as part of the National Cooperative Program on Trophoblast-Maternal Tissue Interactions.

Present address for E.L.L.: Ordway Research Institute, Albany, New York 12208.

Abbreviations: ADAM, A disintegrin and metalloprotease; LIF, leukemia inhibitory factor; MMP, matrix metalloprotease; MT-MMP, membrane-type matrix metalloprotease; P4, progesterone; PG, prostaglandin; PKC, protein kinase C; PMA, phorbol-12 myristate 13-acetate; TACE, tumor necrosis factor {alpha} converting enzyme; TAPI, TNF{alpha} protease inhibitor; TIMP, tissue inhibitor of metalloprotease; TNFR, TNF receptor.

Received March 29, 2004.

Accepted for publication May 6, 2004.


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