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Reproductive Endocrinology |
Department of Obstetrics and Gynecology, New York University Medical Center, New York, New York 10016
Address all correspondence and requests for reprints to: Frederick Schatz, Department of Obstetrics and Gynecology, New York University Medical Center, 550 First Avenue, New York, New York 10016.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Progestin-modulated DZ of cultured human endometrial stromal cells (HESCs) involves changes that reduce ECM-degrading potential. Accordingly, there is coordinate inhibition in the expression of the matrix metalloproteinase (MMP) stromelysin-1 (MMP-3) (3, 4), which degrades several ECM components and can activate other MMP zymogens (5), with the plasminogen activators (PAs) (6, 7), which interact with the MMPs to carry out efficient proteolysis of ECM components (5, 8). Conversely, expression of the potent PA inhibitor-1 (PAI-1) is elevated (7, 9, 10). Consistent with the differential actions of ovarian steroids in vivo whereby E2 priming elevates human endometrial progesterone receptor levels (11), E2 is ineffective alone, but augments the progestin-mediated effects in the HESCs on the expression of MMP-3 (3), the PAs (7), and PAI-1 (7, 10).
Recent work suggested that a model based on in vitro decidualized stromal cells (12) was well-suited to study menstruation-related changes elicited by a decline in circulating ovarian steroids. Thus, after HESCs were incubated with E2 + progestin, the antiprogestin RU 486 (mifepristone) markedly reversed the expression of progestin-enhanced tissue factor (13) and PAI-1 (14) and progestin-inhibited PAs (14). The purpose of the current study was to further explore the utility of cultured HESCs as a menstruation model by comparing steroid withdrawal effects of RU 486 on secreted levels of MMP-3 and PRL. Degradation of the endometrial ECM as mediated by MMP-3 is expected to elicit menstruation-associated sloughing of the functional layer. Moreover, specific targeting by MMP-3 of the endometrial microvasculature ECM should enhance capillary fragility and promote bleeding. These processes are relevant to the clinical administration of RU 486, which leads to endometrial ECM degradation and hemorrhage (15, 16). By contrast, endometrial PRL is a classical DZ marker that does not appear to play a role in menstruation (17). Because endometrial-derived MMP-3 and PRL differ in their relationship to menstruation, which is initiated in response to withdrawal of circulating ovarian steroids, we hypothesize that differential expression of these endpoints will result from removal of such steroids from cultured HESCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After obtaining informed consent, specimens of predecidualized cycling endometria from follicular and luteal phases were derived from patients undergoing hysterectomy for myomas and were transported to a sterile laminar flow hood. A small portion was formalin-fixed for future dating by the histological criteria of Noyes et al. (18). The remainder of each specimen was trimmed and minced in MEM containing 1% of an antibiotic-antimycotic mixture (GIBCO, Grand Island, NY), then digested for 1.5 h at 38 C with type I collagenase (Worthington Biochemical Corp., Freehold, NJ) in basal medium (BM) [a phenol red-free 1:1 vol/vol mix of DMEM (Gibco, Grand Island, NY) and Ham’s F-12 (Flow Laboratories, Rockville, MD), with 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL fungizone] supplemented with 10% charcoal-stripped calf serum (10% SCS). Stromal cells were isolated from the endometrial digestate as previously described (19).
Experimental culture conditions
Parallel cultures of stromal cells were grown to confluence (3–4 x 104 cells/cm2) in a 37 C, 95% air:5% CO2 incubator in BM + 10% SCS, renewing the medium every 4 days.
Steroid specificity
The experimental period was initiated in fresh BM + 10% SCS containing either vehicle control (0.1% ethanol), E2, medroxyprogesterone acetate (MPA), dexamethasone (Dex), E2 + MPA, E2 + Dex, E2 + MPA + RU 486, or E2 + Dex + RU 486. After 5 days in the incubator, the conditioned medium was collected and centrifuged, and the supernatants stored at -70 C for MMP-3 and PRL analysis (see below). The cells of one set of dishes were harvested by scraping with a rubber spatula, then pelleted by centrifugation and frozen for later analysis of DNA and protein content. Fresh corresponding medium was added to the remaining two sets of cultures. These were returned to the incubator, and the procedure repeated for additional 5-day intervals.
RU 486 reversal studies
Using the 37 C incubator, confluent stromal cell cultures were exposed to BM + 10% SCS with 10-8 mol/L E2 and 10-7 mol/L MPA, replacing the medium every 3–4 days. After 10 days, the cultures were washed twice with BM + 10% SCS, and steroid withdrawal was routinely carried out for 4 days in BM + 10% SCS containing either vehicle control or 10-6 M RU 486, while parallel cultures were maintained in 10-8 mol/L E2 + 10-7 mol/L MPA. In some experiments this steroid withdrawal paradigm was carried out for additional 4- and 8-day intervals, whereas in one experiment onapristone (Schering, Berlin, Germany) was substituted for RU 486. The experiments were terminated as described above by collecting the conditioned medium for MMP-3 and PRL determinations and harvesting the cells.
Immunoblot analysis for the presence of MMP-3
Cell-conditioned medium was mixed with Laemmli sample buffer containing 5% 2-mercaptoethanol, subjected to 7% PAGE, then electrotransferred onto nitrocellulose. After blocking nonspecific sites with 5% powdered low fat milk, the blots were incubated with a specific anti-MMP-3 rabbit polyclonal antibody, generously supplied by Dr. M. Lark (Merck Research Laboratories, Rahway, NJ). The blots were then washed and exposed to [125I] protein-A (200,000 cpm/ml) for 30 min. After repeat washing and autoradiography, the effects of the various treatments on relative levels of MMP-3 were assessed by densitometry.
PRL assay
The stromal cell culture-derived conditioned media were thawed and assayed for immunoreactive PRL using a double antibody kit according to the manufacturer’s specifications (Amersham, Arlington Heights, IL).
Protein and DNA assay
The protein content of the cell pellets was determined using a modified Bradford assay (Bio-Rad Labs., Hercules, CA). The total DNA content was determined as previously described (3).
Northern analysis for the presence of MMP-3 messenger RNA (mRNA)
Total RNA was extracted from cultured stromal cells by a guanidinium thiocyanate-chloroform method (RNAzol-B, Cinna Biotecx Laboratories, Houston, TX). Approximately 25 µg total RNA from each of the experimental cultures and molecular weight RNA standards (Boehringer-Mannheim, Indianapolis, IN) were separated on a 1% agarose gel containing 2.2 mol/L formaldehyde, then transferred to a nylon membrane. Levels of MMP-3 mRNA were detected with a probe generously supplied by Dr. N. Hutchinson (Merck Research Laboratories), which was labeled with [32P]deoxy-cytidine 5'-triphosphate to high specific activity by random priming with a Boehringer-Mannheim kit. Hybridization was performed by standard methods, as previously described (13), and the washed filters were exposed to Kodak XAR film (Eastman Kodak, Rochester, NY). Signals were evaluated by densitometric scanning of autoradiograms. To standardize total RNA loads, the membranes were stripped and reprobed with a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe as described previously (13).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immunoblot depicted in Fig. 1
reveals that
E2 + MPA-elicited inhibition of stromal cell-secreted MMP-3
is progestin regulated. Thus, although MPA is a weak glucocorticoid,
the strong glucocorticoid Dex failed to affect secreted MMP-3 levels
whether added alone, or in sharp contrast to the results for MPA, with
E2. Moreover, the antiprogestin RU 486, which can act as an
antiglucorticoid at high concentrations (16), counteracted most of the
E2 + MPA-inhibited MMP-3 output, indicating that RU 486 is
an antiprogestin in the HESCs. The apparent lack of inhibitory response
to MPA alone in this cell preparation reflects the need for
coadministered estrogen to maintain adequate progesterone receptor
levels (11, 20).
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Parallel HESC cultures were incubated with 10-8 mol/L
E2 + 10-7 mol/L MPA, then either continued in
E2 + MPA or E2 alone, or subjected to steroid
withdrawal in medium containing 0.1% ethanol (vehicle control) or
10-6 mol/L RU 486. Figure 2 displays steady
state MMP-3 mRNA levels for the withdrawal intervals of 0–4 and 8–12
days. After 0–4 days, a faint 2.1-kilobase band corresponding to MMP-3
mRNA (3, 21) was detected in HESCs maintained in E2 + MPA.
Its levels were enhanced by the change to control medium and markedly
enhanced in medium containing RU 486. At the end of the 8- to 12-day
interval, continuous exposure to E2 + MPA had virtually
eliminated the appearance of MMP-3 mRNA. Consistent with removal of
residual progestin from the culture medium, up-regulation of MMP-3 mRNA
levels elicited by withdrawal to control medium was now equivalent to
that produced by RU 486. As expected from the progesterone
receptor-augmenting action of E2, the switch to
E2 alone was almost as effective as E2 + MPA in
suppressing MMP-3 levels over the initial 0- to 4-day withdrawal
interval. However, somewhat less inhibition was seen as residual
progestin was removed by 8–12 days. Densitometric evaluation of the
effects of 0–4 days of steroid withdrawal on MMP-3 mRNA levels in
cultured HESCs derived from three endometrial specimens (Fig. 3) shows that RU 486 was about 10-fold more effective
than withdrawal to control medium in reversing E2 +
MPA-elicited inhibition. Although the results shown in Figs. 2 and 3
were obtained with 10-fold excess RU 486 compared with MPA, RU 486
added at twice the MPA concentration in one experiment also effectively
reversed the E2 + MPA-elicited inhibition (results not
shown).
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After confluent HESC cultures were incubated with E2 +
MPA for 10 days, immunoblot analysis was used to measure MMP-3 levels
in the medium of parallel cultures maintained in E2 + MPA
or subjected to steroid withdrawal. Consistent with steroid
withdrawal-elicited increases in MMP-3 mRNA levels (Figs. 2
and 3), the
autoradiogram of Fig. 4 indicates that compared with
cultures maintained in E2 + MPA, secreted MMP-3 protein
levels increased significantly after 0–4 days of steroid withdrawal to
control medium, with much greater enhancement resulting when RU 486 was
added alone or together with E2 + MPA. After 4–8 days, the
withdrawal effects of control medium began to approach those of RU 486
in enhancing secreted MMP-3 levels. Onapristone, an antiprogestin with
fewer antiglucorticoid effects than RU 486 (22), was found to be
equivalent to an equimolar concentration of RU 486 in reversing
E2 + MPA-mediated inhibition of secreted MMP-3 levels in
the one stromal cell preparation studied (results not shown).
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Cultured stromal cells derived from seven endometrial specimens
were incubated for 10 days with E2 + MPA followed by 4 days
of steroid withdrawal. Figure 5 compares MMP-3 levels
measured by immunoblot analysis, and PRL measured by RIA, in aliquots
of the HESC-conditioned medium during the withdrawal period. RU 486
markedly reversed E2 + MPA-inhibited effects on MMP-3
output in each of the seven stromal cell preparations (range, 5 to
75-fold) resulting in a statistically significant mean increase in
secreted MMP-3 levels of about 10-fold (P < 0.009).
HESC-secreted PRL levels are reported to be essentially undetectable
under basal conditions, but are readily detectable in response to
progestins, and further enhanced by E2 + progestins (23, 24). Despite this progestin dependence, and unlike the effects on
secreted MMP-3 levels, RU 486 did not exert consistent effects on PRL
output among the seven experiments. Accordingly, the averaged results
show no significant differences in PRL levels in parallel cultures
maintained in E2 + MPA or withdrawn to either control or RU
486-containing medium.
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Figure 6 shows immunoblot analysis for the presence of MMP-3 in
HESC-conditioned medium during incubation in a defined medium (DM)
[described in (3)] containing either vehicle (control), E2, MPA, E2
+ MPA, interleukin-1ß (IL-1ß), or steroids + IL-1ß. In the
incubations with steroids alone, MPA, but not E2, reduced secreted
levels of MMP-3 (50,000 mw), whereas, augmented inhibition occurred in
response to E2 + MPA. By contrast, IL-1ß markedly elevated MMP-3
levels whether added alone or with the steroids. The stimulatory
effects of IL-1ß not only counteracted E2 + MPA-elicited inhibition
of secreted MMP-3 levels, but produced a further increase above control
levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In nonfertile human menstrual cycles, declining circulating ovarian steroid levels trigger protease-mediated degradation of the endometrial ECM, leading to shrinkage of the stromal compartment, then sloughing and expulsion of the functional endometrial layer in the menstrual fluid (27). A consequence of their widespread distribution in the premenstrual endometrium and high concentration at specific perivascular sites is that decidual cells are well situated to regulate menstrual events. In situ hybridization of human endometrial sections provided evidence to support such a role for decidual cell-derived MMP-3. Thus, MMP-3 mRNA was localized to the stroma of human premenstrual endometrium where its levels were in low abundance during the progesterone-dominated luteal phase, but increased concomitant with estrogen and progesterone withdrawal leading to menstruation (28).
Results presented in this study complement those of other reports to suggest a mechanism by which the actions of RU 486-elicited steroid withdrawal effects on the decidualized HESCs to promote endometrial ECM degradation and bleeding of menstruation. Thus, synergism of RU 486-enhanced MMP-3 activity with RU 486-increased PA activity in decidualized stromal cells (14) would accelerate endometrial sloughing. Moreover, MMP-3-mediated degradation of the perivascular ECM would compromise the structural integrity of endometrial microvessels. Predictably, the resultant increase in capillary fragility would exacerbate bleeding initiated by both tPA, the primary mediator of fibrinolysis (8), as well as the procoagulant tissue factor, the primary mediator of hemostasis via fibrin generation (29). Thus, expression of tissue type PA (tPA) is enhanced (14), and that of tissue factor is inhibited (13) in in vitro decidualized HESCs subjected to RU 486-elicited steroid withdrawal.
RU 486 is an effective abortifacient when administered within 50 postmenstrual days (30), and a contragestive within 72 h of intercourse (31). Because RU 486 interferes with implantation in rodents (32), it could play a similar role in primates. However, the marked endometrial ECM degradation and bleeding that accompany RU 486 use (16) emphasize the utility of in vitro D2 for evaluating clinical aspects of RU 486 use. That use of this model should be extended to measurements of the potent vasoconstrictor endothelin-1, and its inactivator enkephalinase, as suggested by the work of Casey et al. (33). They reported that during the perimenstrual period, the expression of endothelin-1 is increased, whereas that of enkephalinase is inhibited (33). These changes are consistent with the marked vasoconstriction and resulting hypoxia that precedes menstruation.
In contrast to the profound up-regulation in secreted MMP-3
levels triggered by 4 days of RU 486-induced steroid withdrawal, Fig. 5
indicates that parallel aliquots of conditioned medium from HESCs
maintained in E2 + MPA or withdrawn to RU 486 contained
similar PRL levels. In human endometrium, decidual cell-derived PRL
(34) is proposed to play diverse roles in pregnancy including: 1)
osmoregulation of the amniotic fluid (35, 36); 2) modulation of
prostaglandin E synthesis in the human amniochorion (35); and 3)
immunoregulation during implantation (17). Although PRL exerts vascular
effects in rats, the absence of PRL receptors in human endometrial
vasculature argue against the latter as PRL targets. Furthermore, a
survey of the literature turned up no evidence supporting a role for
endometrial PRL in menstruation (17). Previously, Tseng et
al. (37) noted that after PRL expression in HESC monolayers had
been progestin-enhanced, RU 486-elicited steroid withdrawal
superinduced the transcription rate as well as steady state levels of
PRL mRNA. Paradoxical superinduction was similarly observed for
insulin-like growth factor binding protein-1 (37), which mediates
embryo-endometrial interactions (38), but does not appear to play a
role in menstruation.
Steroid responsiveness therefore appears to define two categories of DZ-related markers in HESC cultures. One category includes proteins such as tissue factor, the PAs, PAI-1, and MMP-3. Steroids initiate a change in their expression, and this altered expression is tightly coupled to continued steroid exposure. These proteins are therefore highly sensitive to steroid withdrawal and are postulated to play key roles in menstruation. However, they may also be involved in maintaining hemostasis and in regulating ECM degradation during trophoblast invasion. A second category includes PRL and insulin-like growth factor binding protein-1. Changes in their expression are also steroid initiated. However, continued altered expression is likely regulated by paracrine effectors from trophoblast and placenta. These proteins are postulated to mediate decidual-trophoblast interactions. A recent report showed that several cytokines modulated integrin expression in the cultured HESCs, whereas the cells were refractory to ovarian steroids (39). This may signal the existence of a third category of marker, whose expression by stromal/decidual cells depends solely on paracrine effectors derived from leukocytes, trophoblast, and/or placenta. Interleukin and tumor necrosis factor were recently shown to upregulate the expression of several MMPs, including MMP-3, in the cultured HESCs (40). The observation reported in the current study that IL-1ß abolished E2 + MPA-mediated inhibition of MMP-3 expression in the cultured HESCs suggests that steroid-cytokine interactions are involved in the DZ reaction. Identifying cytokine effectors derived from endometrial cells, leukocytes, trophoblast, and/or placental cells, which act as autocrine/paracrine mediators that induce or inhibit the DZ reaction offers a challenging question for reproductive biologists.
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Footnotes |
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Received April 17, 1996.
Revised July 29, 1996.
Accepted September 9, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and tumor
necrosis factor-. J Clin Endocrinol & Metab 79:530–536.
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