From the Department of Biological Sciences,
University of Delaware, Newark, Delaware 19716 and the
§ Cellular Biochemistry and Biophysics Program,
Sloan-Kettering Institute, Memorial Sloan Kettering Cancer Center,
New York, New York 10021
Received for publication, August 14, 2002, and in revised form, November 8, 2002
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ABSTRACT |
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MUC1 clearance from the uterine epithelial cell
surface is a prerequisite for the creation of an environment conducive
to embryo implantation. In some species, reduced mRNA levels along with metabolic turnover account for loss of MUC1 during the receptive phase throughout the uterine epithelium. In other species, MUC1 is
rapidly lost solely at the site of blastocyst attachment, suggesting the action of a protease. Correlative studies also indicate the presence of soluble forms of MUC1 in cell culture supernatants in
vitro and in bodily fluids in vivo. To characterize
the proteolytic activity mediating MUC1 release, shedding of MUC1 was
analyzed in a human uterine epithelial cell line (HES) that abundantly expresses and readily sheds MUC1. MUC1 release was stimulated by
phorbol 12-myristate 13-acetate and was markedly inhibited by the
synthetic peptide hydroxamate metalloprotease inhibitor, tumor necrosis
factor- Proteolytic removal of the extracellular domain of numerous
transmembrane proteins is responsible for the regulated release of
cytokines, growth factors and their receptors, cell adhesion molecules,
and ectoenzymes (reviewed in Refs. 1-3). This process, referred to as
ectodomain shedding, appears to be essential for mammalian development
(4) and has implicated roles in leukocyte migration, cell-cell
adhesion, and tumor cell proliferation. Ectodomain shedding also is
associated with the progression of several disease processes, including
rheumatoid arthritis (5), other autoinflammatory syndromes (6), and
Alzheimer's disease (Ref. 7; reviewed in Ref. 8). Consequently,
identification of the proteases or "sheddases" involved in the
release of transmembrane proteins should provide insight into the
regulation of key physiological events under normal and pathological
conditions. In this regard, members of the
ADAM1 (for a
disintegrin and metalloprotease)
family of metalloproteases appear to mediate the ectodomain release of
several proteins (reviewed in Refs. 3, 9, and 10). Tumor necrosis
factor (TNF)- Embryo implantation is a highly regulated process that requires both an
attachment competent blastocyst and a receptive uterus. The initial
stage of implantation is mediated by interactions between the apical
surface of the uterine epithelium and the trophectoderm of the
blastocyst. Under most conditions, however, the apical surface of the
uterine epithelium is protected by a thick glycocalyx composed largely
of mucins. MUC1, a transmembrane mucin and an important component of
the glycocalyx, provides a physical barrier to microbial and enzymatic
attack (22, 23). MUC1 exerts its anti-adhesive effect through a large
extracellular domain primarily composed of a series of 20-amino acid
repeats enriched in serine, threonine, and proline residues (24). The
numerous proline residues and extensive O-linked
glycosylation on serine and threonine residues generate a highly
extended and rigid structure that protrudes 200-500 nm into the
pericellular space (25). As a result, a major challenge that the uterus
faces during the receptive phase is to maintain this protective barrier
while permitting blastocyst attachment.
During the receptive phase, and in response to ovarian steroid
hormones, MUC1 expression is reduced throughout the uterine epithelium
in several species (26-29), but is elevated in rabbits and humans (30,
31). In both instances, global changes in MUC1 expression correlate
with changes in MUC1 mRNA levels. However, the presence of the
blastocyst in the rabbit endometrium results in a localized reduction
of MUC1 at the site of implantation (30, 31). Coincidentally, ADAM 9 accumulates at sites of blastocyst attachment (and MUC1 loss) (32),
implicating a role for this ADAM in the implantation process in
rabbits. An in vitro study of cultured human uterine
epithelial cells similarly demonstrated a local loss of MUC1 at the
site of human blastocyst attachment (33). This finding is consistent
with an induced loss of MUC1 at the site of attachment, perhaps
mediated through activation of a cell surface protease.
The aim of the current study was to initially characterize the
mechanism of MUC1 cell surface release. We provide evidence that MUC1
is cleaved from the surface of a human uterine epithelial cell line,
HES, by a protease(s) that is stimulated by phorbol 12-myristate
13-acetate (PMA) and is inhibited by the hydroxamate-based metalloprotease inhibitor, TAPI, and the tissue inhibitor of
metalloproteases (TIMP)-3. We identify TACE as a mediator of
constitutive and phorbol ester-stimulated MUC1 shedding. In addition,
we demonstrate that both TACE and MUC1 are expressed in human uterine
epithelia during the receptive phase in vivo and form a
stable physical association in HES cells in vitro.
Materials--
Phorbol-12 myristate 13-acetate and the furin
inhibitor, decanoyl-RVKR-CMK, were purchased from Calbiochem.
Leupeptin, pepstatin A, and E-64 were obtained from Sigma. TIMP-1,
TIMP-2, TIMP-3, rabbit anti-TACE antibody, and anti-TACE blocking
peptide were obtained from Chemicon. Protein G-Sepharose, Texas
Red-conjugated sheep anti-mouse IgG, and fluorescein isothiocyanate
(FITC)-conjugated donkey anti-rabbit IgG were purchased from Amersham
Biosciences. 4',6-Diamidino-2-phenylindole, dihydrochloride
(DAPI), was purchased from Molecular Probes. Affinity-purified mouse
IgG and rabbit IgG were obtained from Zymed Laboratories
Inc. 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. John Doedens and Dr. Roy Black (Immunex, Seattle, WA).
cDNA Constructs--
The TACE-FLAG cDNA was kindly
provided by Dr. John Doedens. The full-length human MUC1 cDNA, the
generous gift of Dr. Sandra Gendler (Mayo Clinic, Scottsdale, AZ), was
cloned into the expression vector pcDNA3.1 (Invitrogen) to
allow for expression of MUC1.
Reverse Transcription-PCR--
Total RNA was extracted from HES
cells using the RNeasy kit according to instructions from the
manufacturer (Qiagen). Total RNA was extracted from frozen human
endometrial tissue samples using the TRIzol reagent according to
instructions from the manufacturer (Invitrogen). Quantitation and
estimation of purity were performed by measuring the absorbance of each
RNA sample at UV wavelengths of 260 and 280 nm, and integrity was
determined by visual inspection of RNA fractionated by agarose gel
electrophoresis. Reverse transcription was performed using the
Advantage RT-for-PCR kit according to instructions from the
manufacturer (Clontech). PCR was performed using the HotStarTaq Master
Mix kit according to instructions from the manufacturer (Qiagen).
Cell Culture and Shedding Assay--
The human uterine
epithelial cell line, HES, was kindly provided by Dr. Douglas Kniss
(Ohio State University, Columbus, OH). The HES cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented
with 10% (v/v) charcoal-stripped fetal bovine serum (HyClone), 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Cells
were seeded on Matrigel-coated (Becton-Dickinson) 24-well tissue
culture plates (Costar) and maintained as described until cells reached
70-80% confluence. The cells then were serum-starved for 24 h
prior to the beginning of treatment. At the time of treatment, culture
medium was replaced with fresh serum-free medium in the presence or
absence of PMA (1 µM) and leupeptin (10 µM), pepstatin A (10 µM), E-64 (10 µM), TAPI (100 µM), TIMP-1 (20 µg/ml),
TIMP-2 (20 µg/ml), TIMP-3 (20 µg/ml), or the appropriate vehicle
control. Following a 1-h incubation, the cells were examined by phase
microscopy for survival and morphology, and cell lysates and culture
supernatants were harvested for Western blot analysis. In all cases,
cell viability exceeded 95% by the trypan blue exclusion assay.
Wild-type EC-4 and tace Sample Preparation--
The culture supernatants were
transferred into 12 × 75-mm glass culture tubes (Fisher). Fifty
µg of fetal bovine serum protein was added to each sample as a
carrier prior to the addition of 0.2 volume of 50% (w/v)
trichloroacetic acid. The samples were allowed to precipitate at
4 °C for 24 h, followed by centrifugation at 4 °C for 20 min
at 3000 × g. Pellets were rinsed with acetone and
immediately centrifuged, and the supernatants were removed. The pellets
were allowed to dry at room temperature and were resuspended in 25 µl
of sample extraction buffer (SEB) (0.05 M Tris, pH 7.0, 8 M urea, 1.0% (w/v) SDS, 0.01% (w/v) phenylmethylsulfonyl
fluoride, and 1.0% (v/v)
Immunoprecipitation assays were performed as described previously (37).
Briefly, following treatment with PMA or vehicle control, cells were
lysed in 0.5% (v/v) Nonidet P-40 in D-PBS containing
protease inhibitor mixture set III (PICSIII from Calbiochem). The
pre-cleared cell lysates and culture supernatants were incubated overnight at 4 °C by constant rotary motion with rabbit polyclonal antibody CT-1 (38), mouse monoclonal antibody 214D4 (39), or nonimmune
control immunoglobulins. The antigen-antibody complexes then were
incubated at 4 °C for 6 h to overnight with a 50% (v/v) slurry
of protein G-Sepharose. The pellet resin was washed three times with
0.5% (v/v) Nonidet P-40 in D-PBS by resuspension, and centrifugation at 10,000 × g for 3 min followed three
washes with D-PBS alone. The immunoprecipitated MUC1 was
solubilized from the resin by boiling for 3 min in the presence of SEB
and Laemmli sample buffer. Samples were then analyzed by SDS-PAGE.
SDS-PAGE and Detection of MUC1 Protein--
Cell-associated and
culture supernatant protein samples were separated by SDS-PAGE with a
5% (w/v) Laemmli stacking gel and a 10% (w/v) Porzio and Pearson
resolving gel (35, 40). Briefly, the separated proteins were
transferred to Schleicher & Schuell Protran®
nitrocellulose (Intermountain Scientific) at 4 °C for 5 h at 40 V. The blots were blocked for at least 6 h at 4 °C, with 3% (w/v) bovine serum albumin (BSA) (Sigma) in
D-PBS plus 0.1% (v/v) Tween 20 (PBS-T) and
then incubated overnight at 4 °C with the primary antibody, 214D4,
at a final dilution of 1:10,000 in PBS-T containing 3% (w/v) BSA. The
blots were rinsed three times for 10 min each in PBS-T and then
incubated for 2 h at 4 °C, with horseradish
peroxidase-conjugated sheep-anti-mouse IgG (Jackson ImmunoResearch) at
a final dilution of 1:200,000 in PBS-T containing 3% (w/v) BSA.
Following three additional 10-min rinses at room temperature in PBS-T,
MUC1 was detected by the ECL system (Pierce) as described by the
manufacturer. Signal intensities were measured with the one-dimensional
Multi Alpha Imager program (Alpha Innotech). Statistical analyses were
performed using one-way analysis of variance and the Tukey-Kramer
multiple comparisons test (GraphPad InStat program).
Immunofluorescence--
Endometrial specimens were obtained
during routine fertility evaluations of healthy, regularly cycling
women between the ages of 18 and 45 years. Samples were dated
histologically by an experienced gynecological pathologist and by serum
hormone profiles. Samples were frozen immediately in liquid nitrogen
and embedded in OCT cryoprotectant (Baxter) for cryosectioning. The use
of human subjects was approved by the Institutional Review Board of the
University of Delaware.
Frozen mid-luteal human endometrial sections (8 µm) were fixed for 10 min at room temperature in methanol and rehydrated in D-PBS
for 10 min with one change of buffer. Sections were incubated at
37 °C for 1 h with anti-TACE polyclonal antibody diluted to 40 or 1 ng/µl in D-PBS. Preincubation of the anti-TACE
antibody with the TACE peptide for 30 min at 37 °C and nonimmune
rabbit IgG were used as a negative controls. Samples were
rinsed three times for 5 min in D-PBS at room temperature
and incubated at 37 °C for 40 min with fluorescein isothiocyanate
(FITC)-conjugated donkey anti-rabbit IgG diluted 1:10 in
D-PBS. Following three additional 5-min rinses in
D-PBS, samples were incubated for 1 h at 37 °C with
anti-MUC1 monoclonal antibody, 214D4, diluted 1:1 in D-PBS
or nonimmune mouse IgG. Sections were rinsed three times for 5 min in
D-PBS at room temperature and incubated at 37 °C for 40 min with Texas Red-conjugated sheep anti-mouse IgG diluted 1:10 in
D-PBS and DAPI. All samples were mounted in
glycerol:D-PBS containing 0.01% (w/v)
p-phenylenediamine to prevent fading and photographed with a
Zeiss LSM 510 multi-photon confocal microscope.
MUC1 Release Is Rapidly Stimulated by PMA--
Shedding of various
cell surface proteins has been shown to be stimulated by phorbol
esters, such as PMA (20, 41-43), presumably through activation of
metalloproteolytic sheddases. Therefore, we tested PMA for induction of
MUC1 release from HES cells. HES cells constitutively release MUC1, and
as shown in Fig. 1, A
(upper panel) and B, following PMA stimulation,
release was rapidly enhanced at least 6-fold; however, PMA stimulation
did not detectably alter cell-associated MUC1 levels (Fig.
1A, lower panel).
To confirm that the soluble MUC1 observed in the supernatants of HES
cells contained ectodomains only, cell lysates and conditioned medium
from PMA-stimulated and unstimulated cells were immunoprecipitated with
a polyclonal antibody, CT-1, specific for the cytoplasmic domain of
MUC1 or with a monoclonal antibody, 214D4, specific for the
extracellular domain of MUC1 and then examined by Western blot
analysis, probing the membrane with the monoclonal, ectodomain antibody
(Fig. 2). Concurrent with similar
findings in the HES and other cell lines (37, 44, 45), soluble MUC1
from HES cells was immunoprecipitated with 214D4, but not with CT-1,
indicating that MUC1 in the supernatants of unstimulated and
PMA-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," and that constitutive cell surface release of biotinylated
MUC1, devoid of the cytoplasmic tail, accompanies the gradual
disappearance of cell-associated, biotinylated MUC1, immunoprecipitable
with both ectodomain and cytoplasmic domain MUC1 antibodies (37). 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 following PMA treatment is the result of increased release, rather than membrane blebbing.
A Metalloprotease Is Responsible for Shedding of MUC1--
To
initially characterize the activity mediating MUC1 cell surface
release, a series of protease inhibitors were examined for their
ability to modulate MUC1 shedding. Inhibitors of serine (leupeptin),
cysteine (E-64), and aspartyl (pepstatin A) proteases had no effect on
constitutive or PMA-stimulated release of MUC1 (Table
I). In contrast, the hydroxamate-based
metalloprotease inhibitor, TAPI, completely inhibited PMA-stimulated
release (Fig. 3A) and also
substantially diminished constitutive MUC1 release, an effect that was
more readily observable at later time points (data not shown).
To further characterize the MUC1 sheddase(s), we examined the ability
of endogenous metalloprotease inhibitors, TIMP-1, -2, and -3 (reviewed
in Refs. 46 and 47), to inhibit MUC1 shedding. At concentrations known
to inhibit MMPs (48) and ectodomain release of Her2 (49) and TRANCE
(34), TIMP-1 and TIMP-2 failed to inhibit MUC1 release from HES cells
(Table I). Interestingly, TIMP-2 actually stimulated MUC1
shedding.2 However, TIMP-3,
unique in its ability to inhibit TACE/ADAM 17 (50), significantly
inhibited PMA-enhanced shedding of MUC1 (Fig. 3B).
Prodomain removal during biosynthesis is a prerequisite for protease
activity of catalytically active ADAMs and appears to be mediated by a
furin-type proprotein convertase in the trans-Golgi network
(42, 51). Consequently, inhibition of furin might potentially prevent
prodomain removal of the MUC1 sheddase(s) and subsequently inhibit
activation. In this regard, decanoyl-Arg-Val-Lys-Arg (decanoyl-RVKR), a
furin inhibitor (52), was tested for its ability to modulate MUC1
ectodomain release. Interestingly, decanoyl-RVKR was effective at
inhibiting PMA-induced MUC1 release (Fig. 3C), suggesting
that the MUC1 sheddase(s) is processed by a metalloprotease sensitive
to furin or a furin-like proprotein convertase. Collectively, these
data were consistent with a role for an ADAM(s) mediating MUC1 shedding.
HES Cells Express a Majority of ADAMs with a Catalytic Consensus
Sequence--
To address the potential role of an ADAM(s) in
MUC1-mediated shedding, we determined the expression of ADAMs that are
widely expressed and contain a catalytic consensus sequence in HES
cells and in the receptive phase human endometrium by reverse
transcription-PCR. ADAMs 8, 9, 12, 15, 17, 19, and 33 were expressed at
the mRNA level in the HES cells and in the receptive phase human
endometrium (Table II). However, ADAM 28 only appeared to be expressed in the receptive phase human endometrium.
We did not examine expression of ADAM 10 in the HES cells or in the
endometrium because the MUC1 sheddase(s) was not inhibited by TIMP-1,
and TIMP-1 has been shown to inhibit ADAM 10 activity (53).
MUC1 Is Not Shed by tace ADAMs 9, 12, and 15 Are Not Required for MUC1 Shedding--
To
analyze the importance of other ADAMs in MUC1 shedding, we assessed
MUC1 ectodomain release in primary embryonic fibroblasts from ADAM
9/12/15-deficient mice. The wild-type and ADAM 9/12/15-deficient fibroblasts express comparable levels of MUC1 following transfection with MUC1 cDNA (Fig. 5). Moreover,
both cell lines shed MUC1 to a similar or identical extent, arguing
against a role for these ADAMs in MUC1 ectodomain release.
Expression of MUC1 and TACE in HES Cells--
It was considered
that a stable physical interaction might occur between TACE and MUC1
prior to MUC1 cleavage. MUC1 was immunoprecipitated from HES cells with
either 214D4 or CT-1, which recognize epitopes in the ectodomain and
cytoplasmic domain of MUC1, respectively. Immunoprecipitates then were
examined by Western blot analysis for TACE (Fig.
6). A Jurkat cell lysate served as a
positive control for recognition of TACE. The bands detected most
likely represent precursor, glycosylated, and mature forms of TACE.
Preincubation of the anti-TACE antibody with the peptide to which it
was generated effectively eliminated the appearance of these bands
(data not shown). Thus, regardless of the antibody used to
immunoprecipitate MUC1, TACE was detected in all samples except for the
IgG negative control immunoprecipitates, indicating a physical
association between MUC1 and TACE occurs in HES cells. Interestingly,
PMA stimulation appears to increase the association between TACE and MUC1 in cells that have been immunoprecipitated with CT-1 (Fig. 6,
lanes 5 and 6), whereas this interaction is
diminished following PMA stimulation in cells that have been
immunoprecipitated with 214D4 (Fig. 6, lanes 7 and
8).
Localization of MUC1 and TACE in the Receptive Phase Human
Endometrium--
We next considered whether TACE was expressed
appropriately in the human uterus to participate in MUC1 shedding.
Therefore, TACE and MUC1 expression were examined in tissue sections
from the receptive phase human endometrium by fluorescence microscopy. Consistent with previous findings in other systems (42, 55, 56), TACE
displayed an intracellular and perinuclear distribution in luminal and
glandular uterine epithelial cells with barely detectable staining in
the surrounding stromal cells (Fig. 7, A, D, and J). Notably, TACE is
expressed at the apical aspect of luminal and glandular uterine
epithelial cells, which is the predominant site of MUC1 localization
(Fig. 7, B, E, and H). The staining
pattern for MUC1 was not observed with a nonimmune control antibody
(Fig. 7K), and preincubation of the anti-TACE antibody with
the peptide to which it was generated effectively blocked cellular
staining (Fig. 7G). Consequently, this demonstrated
expression of both TACE and MUC1 in epithelia of the receptive phase
human endometrium.
MUC1 plays a critical role in embryo attachment, bacterial
clearance, and various aspects of tumor progression. A high level of
MUC1 expression effectively inhibits cell-cell (57) and
cell-extracellular matrix (39) adhesion. In addition, a correlation has
been observed between overexpression of MUC1, the metastatic potential
of primary tumors, and poor patient survival (58-60). Interestingly,
several findings indicate the presence of soluble MUC1 fragments in
bodily fluids, implying that proteolytic shedding takes place in
vivo (45, 61). Consistent with these findings, studies conducted with breast cancer cell lines (44) and normal mouse uterine epithelial
cells (38) demonstrate that, during normal metabolic turnover, a
significant portion of released cell surface MUC1 lacks the cytoplasmic
tail. Findings in breast, colon, and pancreatic cancer cells, in
addition to normal mammary and prostate, and in HES uterine epithelial
cells also indicate that an intracellular/metabolic proteolytic
cleavage occurs that separates the extracellular domain from the
transmembrane and cytoplasmic tail domains during assembly (37, 62,
63). The MUC1 heterodimer, consisting of the two proteolytic cleavage
products, remains tightly, but noncovalently, associated at the cell
surface through a SEA module, a domain conserved among heavily
glycosylated proteins and thought to facilitate binding of
glycoproteins to neighboring carbohydrate moieties (64, 65), and cannot
be separated by immunoprecipitation with antibodies to the cytoplasmic
tail (38, 62). Interestingly, the MUC1 heterodimer is SDS-labile but
does not dissociate in the presence of urea or Because of its extreme resistance to externally added proteases (23),
it is unlikely that cell surface MUC1 release is mediated by the
actions of an external protease (23, 62). It is more plausible that
there is an endogenous proteolytic system. The studies conducted to
date suggest that the MUC1 heterodimer is an extremely stable complex
and that dissociation of the intracellular cleavage complex is
unlikely, considering that MUC1 mutant transfectants devoid of the
intracellular cleavage site would release little, if any, MUC1 if the
mechanism of cell surface release were simple dissociation.
Collectively, these observations suggest that released MUC1 lacks the
cytoplasmic tail (37, 44) and that release is catalyzed by a protease
(44, 67). Thus, characterization and identification of activities
mediating MUC1 cell surface release are likely to affect our
understanding of the biological function of MUC1 in several contexts.
This study explored the hypothesis that MUC1 is released from the cell
surface by an enzymatic activity. Based on the protease inhibition
profile of the MUC1 sheddase(s), both constitutive and induced MUC1
cell surface release are mediated by a metalloprotease(s). The phorbol
ester, PMA, stimulated MUC1 ectodomain release. In most cases where
TACE plays a role in ectodomain shedding, it has been in the context of
phorbol ester-stimulated shedding (4, 13-15). Serine, cysteine, and
aspartyl protease inhibitors had no effect on MUC1 ectodomain release;
however, constitutive and PMA-mediated MUC1 shedding were sensitive to
the hydroxamate-based metalloprotease inhibitor, TAPI, indicating the
involvement of a metalloprotease in constitutive and stimulated
shedding of MUC1. Interestingly, TAPI also inhibited constitutive MUC1
shedding from a mammary carcinoma cell line, T47D (data not shown).
Furthermore, prodomain removal by a furin-type pro-protein convertase
appears to be a prerequisite for metalloproteolytic activity of the
ADAMs with a catalytic consensus sequence that has been characterized to date (42, 51, 68, 69). Consistent with this observation, stimulated
MUC1 shedding was reduced by a furin inhibitor. Together with the
insensitivity to various protease inhibitors and the sensitivity to
TAPI and the furin inhibitor, these observations suggest the
involvement of a metalloprotease in MUC1 ectodomain release and
indicate that MUC1 ectodomain release is not mediated by dissociation
of the metabolic cleavage complex in light of the susceptibility of
MUC1 release to inhibition by metalloprotease inhibitors.
The endogenous matrix metalloprotease inhibitors, TIMPs, inhibit all of
the known matrix metalloproteases (MMPs) to a varying extent (reviewed
in Ref. 70). Further studies demonstrate that several membrane-type
(MT)-MMPs are effectively inhibited by TIMP-2 and TIMP-3 (71-73).
Accordingly, the TIMP inhibition profile of the MUC1 sheddase(s)
suggests that it is not a known MMP or membrane-type MMP because
neither constitutive nor stimulated MUC1 release is inhibited by TIMP-1
or TIMP-2. In addition, ADAM 10/KUZ is probably not responsible for
proteolytic processing of MUC1 because it is inhibited by both TIMP-1
and TIMP-3 (53). TIMP-3, however, is highly expressed at the
maternal-fetal interface during human implantation, suggesting a
regulatory role in trophoblast invasion (74, 75). It also inhibits the
metalloproteolytic-dependent shedding of L-selectin (76),
syndecan-1 and -4 (77), IL-6R (78), and pro-TNF- A role for TACE in the processing of putative substrates comes from
analysis of the phenotype of tace Previous findings in rabbits indicate a hormonally and
blastocyst-dependent increase in ADAM 9 expression and MUC1
loss at regions of cell-cell adhesion and fusion in the uterus during implantation (32), suggesting a crucial role for ADAM 9 in this species. Clearance of MUC1 from the surface of human uterine epithelial cells also appears to be a necessary prerequisite for the creation of
an environment conducive to blastocyst attachment. In conjunction with
the identification of MUC1 as a TACE substrate and the establishment of
an interaction between MUC1 and TACE in HES cells in vitro, localization of both MUC1 and TACE was observed in vivo in
luminal and glandular epithelial cells from the receptive phase human endometrium. MUC1 is predominantly found on the luminal and glandular epithelial cell surface on microvilli with only modest intracellular localization, whereas TACE is primarily expressed intracellularly with
moderate cell surface expression apparent on luminal and glandular
epithelial cells. The relatively minor degree of intracellular co-localization of MUC1 and TACE suggests that, if TACE is a MUC1 sheddase in vivo, at least a portion of MUC1 cleavage
probably occurs in an intracellular compartment. Consistent with this
finding, various catalytic ADAMs, including TACE, are localized mainly in intracellular compartments and therefore might be active
intracellularly as well as on the cell surface (42). However, in
vitro cleavage studies indicate that a peptide corresponding to
intracellular metabolic cleavage site of MUC1 is not a putative
substrate for TACE nor are other peptides corresponding to potential
cleavage sites within the SEA module of MUC1 (data not shown),
suggesting that TACE does not mediate cleavage of MUC1 at the metabolic
cleavage site or at additional potential cleavage sites within the SEA module. These putative TACE substrates, however, may not be cleaved well in vitro, implicating associations distal to the
cleavage site as necessary in the regulation of MUC1 shedding.
Correlatively, the EGF domain of L-selectin appears to serve as a
protease recognition motif for phorbol ester-stimulated shedding of
this molecule (43). It also has been suggested that interactions
with the cytoskeleton might modulate proteolytic activity by shifting
the localization of proteases to membrane domains proximal to their
substrates following activation (2). In this regard, during the period of receptivity in humans and several other species, the fluidity of the
plasma membrane increases and microvilli on the apical surface of the
luminal epithelium become shorter, more blunted, and irregular (82,
83). Thus, the uterine morphological changes that accompany generation
of a receptive uterine state may temporarily redistribute MUC1 to a
membrane domain where it is more susceptible to cleavage by TACE.
Alternatively, a potential physiologically relevant stimulus of MUC1
shedding might trigger redistribution of TACE and/or MUC1 and thereby
enhance MUC1 shedding.
In conclusion, the current studies provide the initial characterization
of TACE as a candidate MUC1 sheddase in a model of human uterine
epithelia. We demonstrate that MUC1 shedding is stimulated by treatment
with a phorbol ester. Moreover, based on the inhibition profile of the
sheddase(s), both constitutive and stimulated activities appear to be
mediated by the same or a similar protease. The finding that MUC1 and
TACE form stable associations implies that complexes may exist in
uterine epithelia that are poised for rapid cleavage of MUC1. This may
be of importance in human implantation, where local breaching of the
MUC1 barrier by the blastocyst is likely. It remains possible that
additional proteases may cleave MUC1 in vivo. Thus,
determining whether TACE or a related ADAM(s) mediates cell surface
release of MUC1 provides a new venue for the study of MUC1 biology and
potential therapeutic targets for MUC1-dependent processes.
protease inhibitor (TAPI), as well as by an
endogenous inhibitor of matrix metalloproteases, tissue inhibitor of
metalloproteases (TIMP)-3. These characteristics along with studies
conducted with cell lines genetically deficient in various ADAMs (for
a disintegrin and
metalloprotease) identified tumor necrosis factor-
converting enzyme (TACE)/ADAM 17 as a MUC1 sheddase. Furthermore, both
TACE and MUC1 were expressed in human uterine epithelia during the
receptive phase, and co-immunoprecipitation experiments revealed a
physical interaction between TACE and MUC1 in HES cells. These
studies establish a proteolytic mechanism for MUC1 clearance
from a human uterine epithelial cell line and identify TACE as a MUC1 sheddase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
converting enzyme (TACE)/ADAM 17, initially identified
as the catalytic activity responsible for proteolytic processing of
TNF-
(11, 12), also is involved in or has been implicated in the ectodomain release of transforming growth factor-
,
L-selectin, p75 TNF receptor (p75 TNFR) (4),
-amyloid precursor
protein (
APP) (13), p55 TNFR, interleukin-1 receptor (IL-1R) II
(14), erbB4/HER4 (15), the Notch1 receptor (16), IL-6R (17), growth hormone-binding protein (18), cellular prion protein (19), and
fractalkine (20, 21). Moreover, mice that are genetically deficient in
catalytically active TACE display an embryonic lethal phenotype (4),
which underscores the importance of TACE-mediated shedding in
vivo and suggests an essential role for ADAMs in protein ectodomain release. In light of these findings and others, numerous in vitro studies attribute proteolytic processing of
membrane-anchored proteins to one or more ADAMs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Zn/
Zn EC-2 murine fibroblasts
(14) were kindly provided by Dr. John Doedens and Dr. Roy Black and
were cultured in DMEM/F-12 (Invitrogen) supplemented with 1% (v/v) fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 100 µg/ml streptomycin. Primary wild-type and ADAM 9/12/15-null mouse
embryonic fibroblasts (34) were cultured in DMEM supplemented with 10%
(v/v) fetal calf serum, 1% (v/v) nonessential amino acids, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were electroporated using a Bio-Rad GenePulser II with a capacitance extender in 0.4-cm cuvettes at 270 V and 950 microfarads. Five million
cells were used for each electroporation assay. Cells were transfected
with 50 µg of a MUC1 expression plasmid or with empty vector
cDNA. For co-transfection assays, tace
Zn/
Zn EC-2
cells were electroporated with MUC1 and TACE or empty vector cDNA.
Following electroporation, cells were transferred to six-well plates
and allowed to recover overnight in complete growth medium at 37 °C.
At the time of treatment, culture medium was replaced with fresh
serum-free medium in the presence or the absence of PMA (50 ng/ml) and
TAPI (50 µM), or the appropriate vehicle control. Following a 2-h incubation, the cells were examined by phase microscopy for survival and morphology, and cell lysates and culture supernatants were harvested for Western blot analysis.
-mercaptoethanol) and 25 µl of Laemmli
sample buffer (35) and stored at
20 °C. Following collection of
the culture supernatants, cells were rinsed once with Dulbecco's
phosphate-buffered saline (D-PBS), solubilized with SEB,
and stored at
20 °C. Cell-associated protein concentrations were
determined by the method of Lowry (36).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PMA stimulates release of MUC1 from HES
uterine epithelial cells. A, Western blot analysis of
MUC1 expression in cell lysates (lower panel) and culture
supernatants (upper panel) from HES cells treated with PMA
or with vehicle (dimethyl sulfoxide) for 1 h at 37 °C. 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 detected below 205 kDa is the result
of nonspecific recognition by the secondary antibody. B,
shed MUC1 was quantified by densitometric analysis and is expressed as
a percentage of MUC1 released by cells treated with vehicle alone.
Results represent the averages ± S.D. of three or four
independent samples. **, p < 0.01 relative to the
corresponding vehicle control.
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Fig. 2.
MUC1 released from the surface of HES uterine
epithelial cells is devoid of a cytoplasmic tail. A,
schematic representation of MUC1 indicating regions of recognition by
214D4 and CT-1 antibodies as well as the potential site of cleavage.
B, HES cells were treated with PMA or vehicle for 1 h
at 37 °C. MUC1 then was immunoprecipitated from culture supernatants
(upper panel) and cell lysates (lower panel) with
the polyclonal antibody, CT-1 (lanes 5 and 6),
with 214D4 (lanes 7 and 8), or nonimmune rabbit
and mouse control IgG (lanes 1-4). Subsequent Western blot
analysis of the immunoprecipitates was performed with 214D4. This
experiment was repeated three times with similar results.
Protease inhibitor insensitivity of constitutive and PMA-stimulated
MUC1 shedding
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Fig. 3.
PMA-induced MUC1 release is sensitive to the
metalloprotease inhibitor, TAPI, the endogenous metalloprotease
inhibitor, TIMP-3, and the furin inhibitor, decanoyl-RVKR.
Following pretreatment with TAPI, TIMP-3, or decanoyl-RVKR for 1 h
at 37 °C, HES cells were treated with PMA or vehicle in the presence
or the absence of TAPI (A), TIMP-3 (B), or
decanoyl-RVKR (C) for 1 h at 37 °C. MUC1 recovered
from culture supernatants was then examined by Western blot analysis.
Experiments were performed in triplicate and repeated three times with
similar results.
ADAM PCR profile in HES cells and in the receptive phase endometrium
Zn/
Zn EC-2
Cells--
Isolation of cells from ADAM-deficient mice has permitted
direct evaluation of proteases and their potential substrates (4, 13-16, 20, 21, 34, 54). Wild-type EC-4 and tace
Zn/
Zn
EC-2 cells are immortalized embryonic fibroblasts derived from wild-type and tace
Zn/
Zn mice (4). We utilized these
cell lines to assess the importance of TACE in MUC1 ectodomain release.
Neither the wild-type EC-4 nor the tace
Zn/
Zn EC-2
cells express MUC1 endogenously. Therefore, MUC1 shedding was examined
following electroporation of these cells with MUC1 cDNA or empty
vector. Transfected EC-4 cells behaved similarly to HES cells with
respect to constitutive and PMA-induced release of MUC1 (Fig.
4A). Furthermore,
PMA-stimulated MUC1 shedding was sensitive to inhibition by TAPI. In
contrast, constitutive and PMA-stimulated MUC1 shedding were completely
abolished in the TACE-deficient EC-2 cells (Fig. 4B),
although the levels of cell-associated MUC1 were identical in the two
cell populations, demonstrating that TACE is the predominant MUC1
sheddase in this system for constitutive as well as stimulated MUC1
release. To confirm that the lack of MUC1 shedding in the
tace
Zn/
Zn EC-2 cells was the result of the absence of
TACE, we co-transfected the TACE-deficient fibroblasts with MUC1 and
TACE cDNA. As shown in Fig. 4C, constitutive and
PMA-stimulated MUC1 shedding were restored in the co-transfectants.
Taken together, these results demonstrate the critical importance of
TACE in MUC1 shedding in this system.
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Fig. 4.
MUC1 shedding does not occur in
tace Zn/
Zn
EC-2 cells. A, embryonic fibroblasts derived from
wild-type (EC-4) mice were transiently transfected either with cDNA
encoding full-length MUC1 (lanes 3-6) or with the vector
control (lanes 1 and 2) and treated with PMA or
vehicle in the presence or absence of TAPI for 2 h. MUC1 in
culture supernatants (upper panel) and cell lysates
(lower panel) then was examined by Western blot analysis.
Experiment was performed in duplicate and repeated three times with
similar results. B, MUC1 shedding from EC-2 embryonic
fibroblasts derived from tace
Zn/
Zn mice was
determined as described for wild-type EC-4 cells in A. C, EC-2 cells were co-transfected with cDNA encoding
full-length MUC1 and TACE (lanes 3 and 4) or
vector controls (lanes 1 and 2). Cells were
treated with PMA or vehicle for 2 h. MUC1 recovered from the
culture supernatants (upper panel) and in the cell lysates
(lower panel) was assessed as described for wild-type EC-4
cells in A.
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Fig. 5.
MUC1 shedding occurs normally in ADAM
9/12/15-deficient primary embryonic fibroblasts. Primary embryonic
fibroblasts isolated from wild-type (wt) or ADAM
9/12/15-deficient mice ( /
) were electroporated either
with cDNA encoding full-length MUC1 (lanes 3 and
4) or with the vector control (lanes 1 and
2) and treated with PMA or vehicle for 2 h. MUC1 in the
culture supernatants (upper panel) and in cell lysates
(lower panel) then was examined by Western blot
analysis.
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Fig. 6.
Co-immunoprecipitation of MUC1 and TACE from
HES cells. HES cells were treated with PMA or vehicle for 1 h. MUC1 then was immunoprecipitated from cell lysates with CT-1
(lanes 5 and 6), with 214D4 (lanes 7 and 8), or with nonimmune control rabbit and mouse IgG
(lanes 1-4). The immunoprecipitates then were examined by
Western blot analysis with an anti-TACE affinity-purified polyclonal
antibody. Jurkat cell lysate was used as a positive control for the
anti-TACE antibody (lane 9).
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Fig. 7.
Immunolocalization of MUC1 and TACE in tissue
sections from the receptive phase human endometrium.
A-C, tissue sections were incubated with polyclonal
anti-TACE and monoclonal anti-MUC1 (214D4) antibodies followed
by incubation with FITC-conjugated goat anti-rabbit and Texas
Red-conjugated donkey anti-mouse secondary antibodies. Nuclear (DAPI)
staining is shown in the merged images in blue in each case.
Luminal epithelial expression of TACE (A), MUC1
(B), and the merged image (C) were visualized by
confocal microscopy. D-F, Glandular epithelial expression
of TACE (D), MUC1 (E), and the merged image
(F) were processed as described in A-C.
G-I, the anti-TACE antibody was preincubated with the
peptide to which it was generated. Tissue sections then were incubated
with the peptide block and the anti-MUC1 antibody followed by
FITC-conjugated goat anti-rabbit and Texas Red-conjugated donkey
anti-mouse secondary antibodies. TACE peptide block (G),
MUC1 (H), and the merged image (I) were processed
as described in A-C. J-L, Tissue sections were
incubated with the anti-TACE antibody and nonimmune mouse IgG followed
by FITC-conjugated goat anti-rabbit and Texas Red-conjugated donkey
anti-mouse secondary antibodies. TACE (J), mouse IgG
(K), and the merged image (L) were processed as
described in A-C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol,
indicating that disulfide bond formation is unlikely to stabilize the
interaction between the two cleavage products (37, 62). Moreover,
studies conducted with the human uterine epithelial cell line, HES,
indicate that elevated temperature, high salt, and low pH also fail to
disrupt the metabolic cleavage complex (37). Further studies
demonstrate that MUC1 is internalized and recycled repeatedly through
the trans-Golgi network from the apical cell surface to
achieve and maintain a high degree of sialylation (66). A single MUC1
molecule cycling ~10 times through the trans-Golgi network
prior to release with the heterodimeric complex presumably surviving
the repeated cycling through endosomal compartments (66). Taken
together, the apparent stability of the MUC1 heterodimer upon exposure
to conditions that might be encountered during recycling or at the cell
surface along with the finding that deletion of the region comprising
the metabolic cleavage site generates mutant transfectants that are
able to release at least as much cell surface MUC1 as the wild-type
transfectants (62) implicates a second cleavage event at an alternate
stage of processing and argues against dissociation of the MUC1
heterodimer at the cell surface. Nevertheless, Parry et al.
(63) have identified an intracellular cleavage site 65 amino acids
upstream of the MUC1 transmembrane domain. Although this study did not
detect additional amino termini distinct from the metabolic cleavage
site, MUC1 was immunoprecipitated from cells with an antibody to a FLAG
sequence incorporated into the MUC1 ectodomain. Thus, if a second
cleavage occurred, the residual membrane-associated fragment with a
potentially different amino terminus would not have been included in
the immunoprecipitates produced by the antibody to the FLAG sequence.
Therefore, these results do not preclude an additional cleavage event
from occurring at an alternate stage of processing.
(50). Moreover,
TIMP-3 is unique in its ability to inhibit TACE (50, 79) and is the
only TIMP found to bind heparan sulfate proteoglycans expressed on the
cell surface (80), which may permit co-localization and interaction
with cell surface metalloproteases. Thus, the sensitivity of stimulated MUC1 release to TIMP-3 alone and the synthetic metalloprotease inhibitor, TAPI, along with the marked stimulation of MUC1 shedding in
response to PMA are consistent with the involvement of an ADAM protease(s), such as TACE, in proteolytic release of MUC1; however, these studies do not exclude additional or alternative proteases.
Zn/
Zn mice and cells
derived from these TACE-deficient mice. Examination of fibroblasts
isolated from tace
Zn/
Zn mice demonstrates that
TGF-
, L-selectin, p75 TNFR, and
-APP shedding is drastically
reduced in comparison to wild-type cells (4, 13). In addition, numerous
putative substrates for TACE have been identified utilizing
TACE-deficient fibroblasts (14-17, 20, 21). Here, we show that both
constitutive and PMA-stimulated MUC1 release by embryonic
fibroblasts derived from tace
Zn/
Zn mice is completely
abolished in comparison to constitutive and stimulated MUC1 shedding
from the corresponding EC-4 wild-type cells. Because the
tace
Zn/
Zn EC-2 cells are an immortalized cell line,
the immortalization process may have inactivated a protease(s)
expressed in normal cell types. Alternatively, the mutation in the TACE
gene may have affected a linked gene; thus, the defect in MUC1 shedding
may not directly be caused by a loss of TACE. However, co-transfection of TACE and MUC1 cDNA into the TACE-deficient EC-2 cells completely restored constitutive as well as PMA-stimulated MUC1 release. In
addition, primary embryonic fibroblasts devoid of ADAMs 9, 12, and 15 shed MUC1 normally when compared with their wild-type counterparts,
eliminating these ADAMs as potential MUC1 sheddases in this system.
Collectively, these results present compelling evidence for TACE as a
MUC1 sheddase. Nevertheless, further studies will be necessary to
address whether TACE mediates MUC1 ectodomain release at the same site
in the HES cells and in wild-type and TACE-deficient fibroblasts.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. John Doedens and Dr. Roy Black for providing the TAPI, TACE cDNA, and EC-2 and EC-4 cell lines. We are indebted to Dr. A. Fujisawa-Sehara for the gift of the ADAM 12-null mice to Dr. Carl P. Blobel. We thank Dr. John Hilkens for the generous gift of the MUC1 antibody, 214D4, Dr. Sandra Gendler for the gift of the MUC1 cDNA, and Dr. Ari Babaknia for providing the human uterine tissue. We are especially grateful to Dr. Kirk Czymmek for assistance with the Multi-photon confocal imaging and to Sharron Kingston and Margie Barrett for their expert secretarial and graphics assistance. We appreciate the helpful comments and critical reading of this manuscript by Dr. Errin Lagow, and we are grateful to members of the Carson and Farach-Carson laboratories for many helpful discussions.
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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.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 302-831-4296; Fax: 302-831-2281; E-mail: dcarson@udel.edu.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M208326200
2 A. Thathiah and D. D. Carson, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
ADAM, a disintegrin
and metalloprotease;
TNF, tumor necrosis factor;
TACE, tumor necrosis
factor convertase;
p75 TNFR, p75 tumor necrosis factor receptor;
APP,
-amyloid precursor protein;
IL-nR, interleukin-n receptor, where n is a number;
PMA, phorbol myristate acetate;
TIMP, tissue inhibitor of metalloprotease;
DMEM, Dulbecco's modified Eagle's medium;
SEB, sample extraction
buffer;
D-PBS, Dulbecco's phosphate-buffered saline;
FITC, fluorescein
isothiocyanate;
DAPI, 4',6-diamidino-2-phenylindole, dihydrochloride;
decanoyl-RVKR, decanoyl-Arg-Val-Lys-Arg;
MMP, matrix metalloprotease;
SEA, sperm protein, enterokinase, and agrin;
TAPI, TNF-
protease
inhibitor.
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