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Endocrinological Oncology |
Departments of Obstetrics and Gynecology, Division of Human Reproduction and Infertility, University of North Carolina, Chapel Hill, North Carolina 27599; Abington Memorial Hospital (S.S.), Abington, Pennsylvania 19001; and Northern Fertility and Reproductive Associates (A.C.), Meadowbrook, Pennsylvania 19046
Address all correspondence and requests for reprints to: Dr. Bruce A. Lessey, Department of Obstetrics and Gynecology, University of North Carolina, MacNider Building, CB#7075, Chapel Hill, North Carolina 27599. E-mail: lessey{at}addor.med.unc.edu
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Abstract |
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2ß1, 3ß1,
6ß4. PCR demonstrates the expected size
fragments of each, although evidence for alternatively spliced forms of
the 2-subunit was noted. Progesterone treatment of
estradiol-primed cells resulted in increased expression of the
1ß1 collagen-laminin receptor and
suppression of the vß3 vitronectin
receptor, two of the cycle-dependent integrins expressed by normal
endometrial epithelium. These data support the use of Ishikawa cells as
an excellent model to study the regulation endometrial integrins and
advance our understanding of hormonally mediated events surrounding
implantation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although numerous endometrial cancer cell lines have been established
and characterized, the Ishikawa cell line appears to be an excellent
model to study normal endometrial epithelium. These cells, first
described by Nashida et al. (10), maintain estrogen (ER) and
progesterone (P) receptors (PR) (10, 11). The PR in these cells is
inducible by estrogen and suppressible by P and is immunologically and
biochemically similar to PR in normal endometrium (12, 13).
Furthermore, this cell line expresses many of the same enzymes and
structural proteins found in normal endometrium (14, 15, 16, 17, 18). We recently
demonstrated the functionality of both ER and PR in Ishikawa cells (13)
by the induction 1ß1, a P-responsive
integrin family normally expressed on glandular epithelium only during
the secretory phase of the menstrual cycle (8, 19).
In the present study we further characterize Ishikawa cells as a model for the study of normal endometrial epithelium, initiating a general survey of integrins in this cell line. This class of cell adhesion molecules has been implicated in normal cellular phenotype and were measured to judge the similarities between Ishikawa cells and normal endometrial epithelium. Furthermore, the role of cycle-dependent integrin expression in reproduction is becoming increasingly relevant to the process of implantation and the functioning of the endometrium (20, 21, 22). The study of the regulation of hormone-dependent events in a suitable cell model of the endometrium will greatly advance our understanding of the factors that favor the establishment of uterine receptivity toward embryo implantation in the human.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ishikawa cells were grown and maintained in MEM containing 5% (vol/vol) nonstripped FCS, 200 nmol/L L-glutamine, and 5% gentamicin. The cells were maintained in 150-cm2 flasks (Costar, Cambridge, MA) in humidified chambers containing 95% air-5% CO2 at a temperature of 37 C. At near confluency, the cells were treated with culture medium with or without added steroid hormones. The time of exposure varied from 1–4 days. The experimental medium was composed of phenol red-free MEM-Ham’s F-12 (1:1, vol/vol) plus 5% charcoal-stripped FCS. The hormones were added from a 1000-fold concentrated stock in 100% ethanol and 17ß-estradiol (E2; 10-8 mol/L) with or without added P (10-6 mol/L). In certain experiments, the antiprogestin RU-486 (10-6 mol/L) was added to cells treated with E2 plus P.
Immunofluorescence and flow cytometry
Immunoperoxidase staining was performed using the specific
monoclonal antibodies listed in Table 1
, as previously
described (23, 24, 25, 26, 27, 28, 29, 30, 31). Ishikawa cells were grown to confluence on glass
coverslips in 12-well plates (Corning Costar Corp., Cambridge, MA).
After no treatment, or treatment with E2 (10-8
mol/L) or E2 plus P (10-6 mol/L), the
coverslips were lightly fixed in 3.7% formaldehyde in
phosphate-buffered saline (PBS; pH 7.4) for 10 min at room temperature,
followed by a 5-min PBS wash, cold methanol for 4 min, and cold acetone
for 1 min before a final PBS wash for 5 min.
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For flow cytometry, Ishikawa cells were detached from culture flasks using trypsin-ethylenediamine tetraacetate for 5–10 min (Life Technologies, Grand Island, NY), then neutralized with an equal volume of 5% FCS and spun at 300 x g for 10 min; the supernatant was discarded, and cells were resuspended in a volume of PBS, pH 7.4, to yield a concentration of 100,000 cells/100 µL. Then, 500-µL aliquots were added to individual polypropylene tubes, spun down, resuspended in 300 µL PBS-4% BSA containing primary mouse antihuman antibody, and incubated for 60 min at 4 C on a shaker. The sample was spun for 10 min at 300 x g, followed by a wash with 500 µL PBS-4% BSA, and again centrifuged at 300 x g for 10 min. The pellet was resuspended in 300 µL PBS-4% BSA and 1:100 diluted FITC-conjugated horse antimouse IgG and incubated for 30 min at 4 C on a shaker in the dark. Two additional wash/spin steps were performed, then the final pellet was resuspended in 500 µL PBS-0.02% propidium iodide (Sigma Chemical Co., St. Louis, MO). The instrumentation used for flow cytometry consisted of a FACScan linked to a Consort 32 computer with Lysis II software, which was used for both data acquisition and data analysis (Becton Dickinson, San Jose, CA). Cells analyzed by flow cytometry were excited by an argon laser emitting 15 mW at 488 nm. Propidium iodide-stained cells were gated out during data collection. Green fluorescence of the remaining live cells was collected by photomultiplier through a 530/30 nm bandpass filter. A logarithmic amplifier was used to compress the resulting fluorescence signal for storage and display on a 4-decade log scale by Lysis II. Subsequent analysis of green fluorescence was used to determine the relative median fluorescence of each sample. This value was defined as follows. Cells labeled with fluorescent second antibody in the absence of primary antibody were used to obtain the arithmetic median of background fluorescence for each set of samples. This value was subtracted from the median fluorescence of each of the other samples in the set. The resulting adjusted median for each treatment condition was then divided by the adjusted median of its control to obtain the relative median fluorescence of the stimulated sample as a multiple of the control.
PCR
Messenger ribonucleic acid was obtained from Ishikawa cells
grown under standard conditions without hormone treatment, using
oligo(deoxythymidine) chromatography and complementary DNA (cDNA)
produced using a reverse transcriptase kit (Invitrogen, San Diego, CA).
Single stranded cDNA was synthesized for amplification using 50 pmol
oligonucleotide primers 16–23 bp in length in a 50-µL reaction
volume (Invitrogen, San Diego, CA). PCR conditions were: denaturation
at 95 C for 1 min, annealing at 60 C for 1.5 min, extension at 72 C for
3 min, followed by the second cycle with extension at 72 C for 10 min.
Oligomer primers for PCR for the 1-, 2-,
3-, 4-, 5-,
6-, and ß4-subunits and the expected sizes
of the PCR products are shown in Table 2. Products of
PCR were separated on 1% agarose gel electrophoresis containing
ethidium bromide and visualized and photographed on a UV light box. The
sizes of the separated products were determined by concomitant
separation of size markers of DNA.
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Cell membrane lysates were obtained from Ishikawa cells treated with no hormones, with E2 (10-8 mol/L) with or without P (10-6 mol/L), or with E2 plus P plus RU486 (10-6 mol/L). Before preparation of membrane protein lysates, cells were incubated for 1 h with 100 µL biotin (10 µg/µL in PBS; Sigma Chemical Co.) and rinsed three times. Membrane extracts were prepared by lysis with Nonidet P-40-containing buffer as previously described (8). The membrane extract was precleared with protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden). Using specific monoclonal antibodies, membrane extract was incubated with primary antibody and rocked for 1 h at 4 C before precipitation with protein A-Sepharose. After five washes, samples were extracted in 1-fold concentrated sample buffer and analyzed by 7.5% SDS-PAGE under nonreducing conditions. Gels were then transferred to nylon membrane. The membrane was blocked with 5% milk (from dehydrate) in tris-buffered saline plus 0.05% tween 20, pH 7.5 incubated with goat antimouse IgG (1:100), washed, and incubated in avidin conjugated to alkaline phosphatase (Dako, Carpenteria, CA) before incubation with ECL chemoluminescence reagent (Amersham, Arlington Heights, IL). Chemoluminescence was developed on x-ray film for 5–10 min before film development.
For Western blot of the ß3-integrin, membrane extract was prepared from Ishikawa cells that had been pretreated for 4 days with E2 (10-8 mol/L) or E2 plus P (10-6 mol/L) or in the presence of E2 plus P plus equimolar RU-486 (10-6 mol/L). The protein concentration was determined by the method of Lowry, and equal amounts of protein were added to 1-fold concentrated sample buffer and analyzed by 7.5% SDS-PAGE under nonreducing conditions as described above. The resulting blots were blocked as described and then incubated with SSA6 supernatant for 1 h at room temperature. The blots were incubated with goat antimouse IgG (1:100) and then developed as described for immunoprecipitation experiments.
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Results |
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2ß1,
3ß1, and 6ß4
(8). Each of these integrins serves as a receptor for either collagen
or laminin, found in basement membranes. As shown in Fig. 1, using flow cytometry performed on Ishikawa cells
shows that these cells expressed the collagen/laminin receptors
subunits 2, 3, 6, and
ß4 compared to the negative control using no primary
antibody (Fig. 1). These cells (in the absence of added hormones)
expressed little if any 4. Untreated Ishikawa cells
expressed both subunits of the vß3
vitronectin receptor. This integrin has been shown to appear on normal
endometrial epithelium at the time of implantation (8, 13, 23).
Photomicrographs of immunofluorescence confirms these findings (Fig. 2, B–H), compared to negative controls (Fig. 2A). Note
that the stromal integrin subunit 5 was not expressed in
these cells (Fig. 2E) as previously reported in normal endometrial
epithelium.
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2,
3, 6, and ß4 messenger
ribonucleic acid transcripts, each with the predicted size (Fig. 3, lanes 1–4, respectively), although two smaller bands
were also visible for the 2-subunit, suggesting
alternatively spliced forms may be expressed in this cell line,
compared to the expected size based on the 2 cDNA (lane
6). The 5-subunit was not detected in Ishikawa cells by
PCR (lane 5).
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). Under these conditions,
Ishikawa cells express little 1, but express the
2ß1- and
3ß1-integrins seen in normal endometrial
epithelium (lanes 2 and 3). The 4ß1- and
5ß1-integrins were not present in these
cells (lanes 4 and 5). In contrast to the 1-integrin
subunit, which was barely detectable (lane 1),
vß3 was strongly expressed (lanes 6 and 7,
respectively).
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, there was a clear induction of the
1-subunit after treatment with E2 plus P,
but none with control medium or E2 treatment alone
(inset). With E2 or P treatment alone (bar
graphs in Fig. 5), there was little effect on 1
compared to the control. If cells were pretreated for prolonged periods
with E2 before P treatment for only 1 day, 1
levels were higher (fourth bar) than those after concomitant
E2 plus P treatment for 4 days (third bar). The
positive effect of E2 plus P was reversed by the addition
of the antiprogestin RU-486.
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and 4, the
vß3-vitronectin receptor appeared to be
expressed in these cells in the absence of hormone treatment. The
expression of vß3 was reduced by
E2 treatment and further suppressed by E2 plus
P, as shown by flow cytometry (Fig. 6A) and Western blot
analysis (Fig. 6B). This suppression of
vß3 was reversed by the addition of the
antiprogestin, RU486. P treatment alone had little or no effect on
ß3 expression (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 vitronectin receptor, is expressed on
endometrial epithelial cells only at the opening of the implantation
window, on postovulatory day 6 (8). It has subsequently been
demonstrated that certain integrins may be useful markers of uterine
receptivity, coexpressed only during the window of implantation (LH
plus 6 to 10) (23). Further, aberrant expression of the
vß3 vitronectin receptor has been
demonstrated in several clinical settings associated with infertility
(6, 7, 8, 32). Complementary expression of integrins on embryonic
epithelial cells has lead to speculation of integrin involvement in the
early cell-cell adhesion events during embryo-endometrial interactions
(33, 34, 35). Although data increasingly support the use of integrins as
markers of uterine receptivity, an understanding of the factors
regulating their expression and their role in implantation remains
incomplete.
This is the first general surveys of integrin expression in an
endometrial cell line compared to normal endometrial epithelium. The
findings described herein reconfirm how similar these cells are to
normal endometrial epithelium. These cells have several constitutively
expressed integrins on their surface, including
2ß1, 3ß1, and
6ß4. Aside from their expression of
various integrin subunits, these cells also appear to respond
appropriately to steroid hormone stimulation (12, 13) and produce other
known endometrial proteins (36). Although many cell types express
similar integrins, and some integrins are present on many different
cell types, it is reassuring to note that this model system so closely
resembles glandular endometrial epithelium. Although endometrial
adenocarcinomas generally conform to the pattern of integrins seen in
normal endometrium, overall integrin expression is reduced with
increasing grade of the tumor (37). As a well differentiated cell line,
Ishikawa cells have retained a more normal phenotype. Using PCR, we
documented what appear to be several alternately spliced forms of the
2-subunit. Although many integrins have been noted to
have such alternative forms, this has not been previously described in
the 2-subunit, and its significance is unknown.
The endometrium appears unique, exhibiting hormonally induced integrin
expression. In normal endometrial epithelium, both 1-
and 4-subunits appear at the time of ovulation, and the
former has been shown to be inducible in vitro by
pretreatment with P (38). With the exception of
4ß1, a fibronectin receptor described in
the secretory phase of normal endometrium (19, 23), the specific
complement of endometrial epithelium integrins is expressed in
toto by Ishikawa cells. The
4ß1-integrin may serve to bind to the
alternatively spliced form of fetal fibronectin (19), although its role
in implantation remains poorly understood. Based on these studies, we
may now begin to better understand the basis for the cycle-dependent
expression of 1ß1 and
vß3 in the endometrial epithelial
cells.
There is little doubt that P is required for the successful
establishment of pregnancy. We (39) and others (40, 41) have shown that
the fall in epithelial PR temporally corresponds to the time of
implantation and the appearance of the
vß3-integrin on endometrial epithelium (8, 42). Unlike the 1-subunit, which is induced by P at the
time of ovulation, the vß3-integrin
appears to be a P-suppressed protein. The loss of PR may signal a
transition from epithelial-mediated events to a stromal-dominant
period, coincident with the later events of implantation. This concept
is supported by recent immunohistochemical studies on endometrium from
women with histologic delay and vß3
expression (42). In that study, endometria from patients with luteal
phase defect and aberrant integrin fail to down-regulate their
epithelial PR in a timely fashion. After successful treatment with
exogenous P or the antiestrogen clomiphene citrate to restore normal
"in phase" histology, we noted the appropriate decline in PR and
the concomitant return of vß3 expression.
Provocative new data using the promoter for the
ß3-subunit in a CAT reporter gene construct also supports
this relationship between steroids and ß3 gene
suppression, which demonstrates that ß3 appears to
normally be suppressed in the endometrium by the sex steroids (43).
In Ishikawa cells, the observed changes in integrins are consonant with
the hypothesis that both 1- and
ß3-subunits are coregulated by P. In the former case,
1 is stimulated by the addition of P to
E2-primed Ishikawa cells. Further, this P-mediated event is
inhibited by addition of the antiprogestin, RU-486 and is augmented by
extending the time of E2 treatment. The Ishikawa cells
maintain a basal level of vß3 expression.
The inhibition of this integrin by P was also dependent on priming with
E2 and is consistent with the shifts in
vß3-integrin in vivo. In the
proliferative and early secretory phases, epithelial
vß3 is absent, whereas epithelial PR and
serum P are both elevated. In Ishikawa cells, E2 and P
mimic the early secretory phase, and addition of the antiprogestin,
RU-486, mimics the midsecretory phase, when
vß3 first appears. In normal endometrium
this is due to the down-regulation of PR; in Ishikawa cells, this is
due to blockade of this receptor. These data demonstrate the usefulness
of such models to study hormonally regulated events in the
endometrium.
In conclusion, we have studied expression of the ß1- and
ß3-integrin family in the well differentiated Ishikawa
endometrial cancer cell line. As previously demonstrated, these cells
maintain both ER and PR (9), with inducible PR identical to normal
endometrial epithelium (13). Ishikawa cells express similar complement
of integrins as their normal epithelial counterpart. The
2-integrin appears to exist in this cells line in
alternatively spliced forms. Like normal endometrial epithelium,
cycle-specific expression of 1 and ß3 also
appears intact. These studies provide objective evidence that the
ß3-subunit of the vß3
vitronectin receptor is a P-suppressed protein and confirms that
1 expression is stimulated by P. The Ishikawa
endometrial adenocarcinoma cell line is an excellent model system for
the study of steroid-mediated events and the cellular mechanisms that
are important to the establishment of a receptive endometrium for
implantation.
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Footnotes |
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Received March 14, 1996.
Revised July 18, 1996.
Revised September 12, 1996.
Accepted September 23, 1996.
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References |
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and common ß subunits. J
Cell Biol. 105:1873–1884.This article has been cited by other articles:
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C K Wang, R S Robinson, A P F Flint, and G E Mann Quantitative analysis of changes in endometrial gland morphology during the bovine oestrous cycle and their association with progesterone levels Reproduction, August 1, 2007; 134(2): 365 - 371. [Abstract] [Full Text] [PDF]
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 B. Mo, A. E. Vendrov, W. A. Palomino, B. R. DuPont, K.B.C. Apparao, and B. A. Lessey ECC-1 Cells: A Well-Differentiated Steroid-Responsive Endometrial Cell Line with Characteristics of Luminal Epithelium Biol Reprod, September 1, 2006; 75(3): 387 - 394. [Abstract] [Full Text] [PDF]
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K.B.C. Apparao, L. P. Lovely, Y. Gui, R. A. Lininger, and B. A. Lessey Elevated Endometrial Androgen Receptor Expression in Women with Polycystic Ovarian Syndrome Biol Reprod, February 1, 2002; 66(2): 297 - 304. [Abstract] [Full Text] [PDF]
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S. Kimmins and L. A. MacLaren Cyclic Modulation of Integrin Expression in Bovine Endometrium Biol Reprod, November 1, 1999; 61(5): 1267 - 1274. [Abstract] [Full Text]
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R.R. Gonzalez, A. Palomino, A. Boric, M. Vega, and L. Devoto A quantitative evaluation of {alpha}1, {alpha}4, {alpha}V and {beta}3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal Hum. Reprod., October 1, 1999; 14(10): 2485 - 2492. [Abstract] [Full Text] [PDF]
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 S. G. Somkuti, L. Yuan, M. A. Fritz, and B. A. Lessey Epidermal Growth Factor and Sex Steroids Dynamically Regulate a Marker of Endometrial Receptivity in Ishikawa Cells J. Clin. Endocrinol. Metab., July 1, 1997; 82(7): 2192 - 2197. [Abstract] [Full Text] [PDF]
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