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Original Studies |
Department of Gynecology and Obstetrics, Stanford University Medical Center and School of Medicine (H.Y.H., Y.W., J.C.I., J.S.K., M.L.P.), Stanford, California 94305; and the Department of Obstetrics and Gynecology, Lin-Kou Medical Center, Chang Gung Memorial Hospital and University School of Medicine (H.Y.H., Y.K.S.), Taipei, Taiwan
Address all correspondence and requests for reprints to: Dr. Hong-Yuan Huang, Department of Gynecology and Obstetrics, Stanford University Medical Center and School of Medicine, Stanford, California 94305.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 92-kDa type IV collagenase, an important enzyme for degradation of the basement membrane (primarily collagen type IV), is crucial for the invasive ability of trophoblast cells (9, 10, 11). The type IV collagenases have been shown to cleave native collagen at a single site into two fragments (12, 13) and are expressed in migrating trophoblasts from outgrowth mouse blastocysts (14). The regulation of type IV collagenase expression may play an integral role in embryo implantation and placentation (15). The naturally occurring specific inhibitors, tissue inhibitors of metalloproteinases (TIMP), of decidual or trophoblast cell origin also have an important physiological role in regulating trophoblast invasion (16). TIMP-1 is a glycoprotein with a molecular mass of 28.5 kDa, which binds in a stoichiometric manner to form a complex with activated interstitial collagenase, stromelysin, and 92-kDa type IV collagenase (17). TIMP-2 is a 21-kDa protein capable of binding to both the latent and activated forms of 72-kDa type IV collagenase (18, 19). TIMP-3, a novel member of the TIMP family, has been shown to have inhibitory activity against stromelysin-1, collagenase-1, and 92-kDa collagenase (20). Studies have demonstrated the expression of TIMP, in mouse uterine wall (21) and hatching blastocysts (14), with attenuation of trophoblast penetration in vitro. Significant expression of TIMP-3 is seen in maternal cells in the area surrounding invading mouse embryonic tissue (22). Simultaneous expression of 92-kDa type IV collagenase and its inhibitors in early human decidua suggests that the activity of 92-kDa type IV collagenase is also regulated by TIMP, which may play an important role in the myometrial invasion of trophoblast cells (23).
Human endometrium is an active site for cytokine production and action (24, 25). Several studies strongly suggest a critical role for autocrine/paracrine cytokines as major local regulators of steroid hormone action (26) and implicate them in the implantation process (27). The presence of the complete interleukin-1 (IL-1) system, including IL-1ß messenger ribonucleic acid (mRNA) expression (28, 29), IL-1 receptor (IL-1R) tI (30, 31, 32), and icIL-1 receptor antagonist (33), has been documented in human endometrium. Other evidence also supports a role for the IL-1 system in human trophoblast physiology. IL-1 was detectable in conditioned medium from trophoblast cultures, with a regulatory effect on hCG and PGE2 production (34, 35), and there is immunoreactive evidence of the IL-1 system in the materno-trophoblast unit (29). IL-1 may also play an intermediary role in trophoblast invasion by regulating trophoblast expression of 92-kDa type IV collagenase (36).
Transforming growth factor-ß (TGFß) is a family of polypeptides with the ability to regulate in vitro differentiation and proliferation of a variety of cell types depending on the microenvironment (37). TGFß is detectable in conditioned medium from first trimester decidua and trophoblast cell cultures and appears to mediate trophoblast invasion (38, 39). Endometrial stromal cell differentiation is essential for implantation of outgrowth blastocysts (40), and the expression of 92-kDa type IV collagenase was shown to be regulated by several cytokines, including IL-1 and TGFß, in many cell types (41, 42, 43, 44). Thus, we hypothesized both IL-1 and TGFß may play a crucial role in embryo implantation at the embryo-maternal interface by regulating stromal cell expression of 92-kDa type IV collagenase, TIMP-1, and TIMP-3, all of which are known to be important in trophoblast invasion. Therefore, we examined the regulation of 92-kDa type IV collagenase, TIMP-1, and TIMP-3 mRNA expression in cultured human stromal cells by IL-1ß and TGFß using quantitative competitive PCR (QC-PCR).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human luteal phase endometrium was obtained from surgical specimens of normally cycling women undergoing hysterectomy for benign reasons, in accordance with the guidelines of the Declaration of Helsinki, after informed consent had been obtained and with approval by the Stanford University human subjects committee. The tissue samples used for this study were histologically normal. Stromal cells were separated from the glandular epithelium after collagenase digestion and were cultured using an established in vitro model, as previously described (31, 45), in 75% DMEM (Life Technologies, Grand Island, NY) and 25% MCDB-105 (Sigma Chemical Co., St. Louis, MO), containing antibiotics, 5 µg/mL insulin (Sigma), and 10% charcoal-stripped FBS (Gimmini, Calabasas, CA). Cultures prepared by this method contained less than 0.1% endometrial epithelial or vascular cells (45).
Hormonal treatment
Stromal cells (1–7 passages) were plated at 2 x 105/well in 24-well culture plates (Falcon, Becton Dickinson, Lincoln Park, NJ) and cultured in standard medium. After confluence (designated day 1), cell cultures were treated with serum-free standard medium supplemented with 10 µg/mL human apo-transferrin (Sigma), 50 µg/mL ascorbic acid (Sigma), 1 µmol/L P (Sigma), 10 nmol/L E (Sigma), 20 ng/mL epidermal growth factor (Sigma), and 1 mg/mL BSA (Irvine, Santa Ana, CA) for 9 consecutive days. Control confluent cells were cultured in the same medium in the absence of E and P. Unless indicated otherwise, standard medium and serum-free medium were renewed every 2–3 days throughout the culture period. Conditioned serum-free standard medium was collected and frozen at -70 C until assayed for endogenous IL-1ß and PRL production.
Dose-response study of recombinant human IL-1ß (rhIL-1ß)
Confluent stromal cells treated with steroid hormones for 9 days were stimulated with rhIL-1ß (1 x 105 IU/µg; Genzyme, Cambridge, MA) in a dose-dependent study (0–1000 IU/mL) for 24 h. As a control for IL-1ß specificity, stromal cells were cultured with serum-free medium in the presence of rhIL-1ß (100 IU/mL) and neutralized with increasing concentrations of anti-IL-1ß monoclonal antibody (Genzyme) in a dose-dependent manner (0–2 µg/mL) for 24 h.
Dose-response study of human TGFß
Cultured stromal cells treated with steroid hormones for 9 days were stimulated with human TGFß1 (0–40 ng/mL; Genzyme) for 24 h in a dose-dependent study. As a control for TGFß1 specificity, stromal cells were cultured with serum-free medium in the presence of TGFß1 (5 ng/mL) and neutralized with anti-TGFß monoclonal antibody (Genzyme) at a concentration of 10 µg/mL for 24 h (46).
RNA analysis
Total RNA was extracted from cultured stromal cells using the guanidinium isothiocyanate method (RNAzol, Tel-Test, Friendswood, TX). The RNA concentration was quantified by measuring optical density with a Spectronic 601 spectrophotometer (Milton Roy Co., Rochester, NY). RNA was diluted to 1 µg/µL for RT-PCR. All experiments were performed a minimum of three times with similar results.
Enzyme-linked immunosorbent assay (ELISA) for IL-1ß and PRL levels in conditioned medium
Conditioned media were collected before the addition of cytokine for the measurement of endogenous produced IL-1ß using an ELISA kit (R&D Systems, Minneapolis, MN) with a detection limit of 1 pg/mL, an intraassay precision of 2.3–3.4%, and an interassay precision of 3.4–7.1%. As a marker of decidualization, PRL in conditioned medium derived from the end of cultures was measured by ELISA (Diagnostic Systems Laboratories, Webster, TX) with a detection limit of 0.14 ng/mL and intra- and interassay coefficients of variation of 5.5–9.0% and 6.6–10.4%, respectively. All samples were assayed in triplicate.
Preparation of oligonucleotide primers for RT-PCR
Specific sequences of oligonucleotide primers for detecting
stromal cell expression of human 92-kDa type IV collagenase (47),
TIMP-1 (48), TIMP-3 (49), and IL-1R tI (50); were obtained from the
GenBank database of the National Center for Biotechnology Information
of the NIH. The corresponding primers were synthesized at the Beckman
Center, Stanford University Medical Center (Stanford, CA). To ensure
that the product detected resulted from amplification of specific
complementary DNA (cDNA) in question rather than contamination of other
cDNAs, all of the primers were designed to span the exon and intron
regions. ß-Actin message was amplified as a control molecule using
primers for the human sequences obtained from Clontech Laboratories
(Palo Alto, CA) (51). To document the presence of an intact IL-1
agonist-receptor system in the cultured stromal cells, the mRNA
expression of IL-1R tI was examined. Human luteal endometrium from
endometrial biopsy specimens of normal cycling women is known to
express all of these transcripts and was used as a positive control to
identify cDNA fragments generated using the various primers. As a
negative control, a defined volume of cultured medium was extracted and
subjected to the same RT-PCR reaction for different specific primers. A
summary of oligonucleotide primer sequences is presented in Table 1
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For RT-PCR, the GenAmp RNA PCR kit (Perkin-Elmer, Foster City, CA) was used. Nineteen microliters of RT-Master Mix for each sample were prepared containing 5 mmol/L MgCl2, 1 x PCR buffer II, 1 mmol/L of each deoxy-NTP, 2.5 µmol/L oligo(deoxythymidine)16, 20 IU ribonuclease inhibitor, and 100 IU Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer). The reactions were started with 1 µg total RNA extracted from stromal cells in a total volume of 20 µL RT-Master Mix and filled into a 0.5-mL thin wall PCR tube (Applied Scientific, South San Francisco, CA). RT-Master Mix in PCR tubes was covered with 50 µL light white mineral oil (Sigma). The RT reaction was carried out in the DNA Thermal Cycler 480 (Perkin-Elmer GeneAmp, PCR Instrument System, Branchburg, NJ) using a program with one 15-min RT cycle at 42 C, followed by 5 min at 99 C, then quenched at 4 C. Products were stored at -20 C until the subsequent PCR.
PCR
Aliquots of the RT products were subjected to PCR in the PCR Master Mix containing 2 mmol/L MgCl2 solution, 1 x PCR buffer II, 2.5 IU Ampli-Taq DNA polymerase (Perkin-Elmer), and corresponding paired primers in a concentration of 0.2 µmol/L to a total volume of 100 µL. PCR was performed simultaneously from a single Master Mix in the different tubes with each primer. PCR cycles were composed of 1 cycle of 95 C for 5 min to denature all proteins, 30 cycles of 45 s at 94 C, 45 s at 55 C, and 60 s at 72 C. The reaction was terminated at 72 C for 5 min and was quenched at 4 C.
Agarose gel electrophoresis
Two percent agarose gel (Life Technologies) electrophoresis was carried out in an H5 electrophoresis chamber. Gels were stained with ethidium bromide (Sigma). Aliquots (25 µL) of each PCR product and dye buffer were analyzed in parallel with a 100-bp DNA ladder (Life Technologies) as a standard. After completion of electrophoresis, the gel blot was analyzed, and photocopies of the blot were printed by UV densitometry (Gel-Doc 1000 system, Bio-Rad Laboratories, Hercules, CA).
QC-PCR
Using an internal standard cDNA for QC-PCR as previously
described (52, 53), quantitative mRNA expression of 92-kDa type IV
collagenase, TIMP-1, and TIMP-3 in cultured stromal cells was
determined. A competitive cDNA fragment was constructed by deletion of
a 277-bp fragment from the 92-kDa type IV collagenase target cDNA to be
detected as illustrated representatively in Fig. 1. The deleted cDNA fragment was
synthesized from 1 µg human endometrial RNA amplified with the 5'-end
original primer and 3'-end competitive primer (listed in Table 1
) and
was purified from 2% agarose gel with an agarose gel DNA extraction
kit (Boehringer Mannheim, Mannheim, Germany). Competitive cDNA
fragments for TIMP-1 and TIMP-3 were constructed by deletion of 104-
and 505-bp fragments, respectively, from the corresponding target cDNA
using same methodology. To establish the equivalence of each target
cDNA to internal standard cDNA used in the QC-PCR, a serial dilution of
competitive cDNA for 92-kDa type IV collagenase added to each PCR
sample and coamplified with target cDNA was illustrated
representatively in Fig. 2. After
completion of RT, a defined amount of competitive cDNA for 92-kDa type
IV collagenase (ranging from 16–32 pg), TIMP-1 (512 pg to 1 ng), and
TIMP-3 (0.5–1 pg), and the corresponding specific target cDNA
coamplified in one reaction with the same primers were used. Aliquots
(25 µL from 30 µL) of each PCR product were electrophoresed on 2%
agarose gel and scanned using UV densitometry. The abilities of
increasing concentrations of IL-1ß and TGFß1 to modulate TIMP-1,
TIMP-3, and 92-kDa type IV collagenase mRNA expression were measured
quantitatively by calculating the ratio of target cDNA to internal
standard cDNA (54).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the ability of IL-1ß to mediate TIMP-1, TIMP-3, and
92-kDa type IV collagenase mRNA expression in stromal cells, confluent
cultures were treated with steroid hormones in serum-free medium for 9
days and cultured for an additional 24 h, as described in
Materials and Methods, in the absence or presence of rhIL-1
ß (10 IU/mL). Figure 3A shows the
positive control of total RNA from normal human luteal endometrial
biopsy amplified with primers for ß-Actin, TIMP-1, TIMP-3, IL-1R tI,
and 92-kDa type IV collagenase and the negative control of conditioned
medium derived from stromal cell cultures amplified with identical
primers in same PCR reaction. Figure 3B demonstrates the
IL-1ß-dependent expression of 92-kDa type IV collagenase mRNA
expression in cultured stromal cells in the presence of 10 IU/mL of
rhIL-1ß.
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To further assess the IL-1ß-mediated regulation of TIMP-1,
TIMP-3, and 92-kDa type IV collagenase mRNA expression in stromal
cells, confluent stromal cells were treated with steroid hormones in
serum-free medium for 9 days and cultured for an additional 24 h,
as described in Materials and Methods, in the absence or
presence of increasing concentrations of rhIL-1ß (0–1000 IU/mL).
Figure 4 shows a ratio of target to
internal standard cDNA documenting a dose dependent up-regulation of
92-kDa type IV collagenase (Fig. 4A) and down-regulation of both TIMP-1
(Fig. 4B) and TIMP-3 (Fig. 4C) mRNA expression with increasing
concentrations of IL-1ß.
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The effect of IL-1ß-mediated regulation of TIMP-1, TIMP-3, and
92-kDa type IV collagenase mRNA expression in stromal cells was
attenuated by anti-IL-1ß antibody in a dose-dependent manner.
Confluent stromal cells were treated with steroid hormones in
serum-free medium for 9 days and cultured for an additional 24 h,
as described in Materials and Methods, in the absence or
presence of rhIL-1ß (100 IU/mL) with increasing concentrations of
anti-IL-1ß antibody (0, 1, and 2 µg/mL). The increase in 92-kDa
type IV collagenase mRNA expression induced by IL-1ß was abolished by
increasing the concentration of anti-IL-1ß antibody (Fig. 5A). The decrease in both TIMP-1 (Fig. 5B) and TIMP-3 (Fig. 5C) mRNA expression in the presence of rhIL-1ß
(100 IU/mL) was reversed with increasing concentrations of anti-IL-1ß
antibody.
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To assess the effect of TGFß1 on the mRNA expression of TIMP-1,
TIMP-3, and 92-kDa type IV collagenase, confluent stromal cells were
treated without steroid hormones (Fig. 6A) and with steroid hormones (Fig. 6B)
for 9 days. Cells cultured with steroids were then treated with TGFß1
(5 ng/mL; Fig. 6C) and rhIL-1ß (100 IU/mL; Fig. 6D) for an additional
24 h, demonstrating that TIMP-1 and TIMP-3 are expressed in cells
cultured with and without steroid hormones. In addition, TGFß1 does
not stimulate 92-kDa type IV collagenase mRNA expression.
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To further describe TGFß1-mediated regulation of TIMP-1 and
TIMP-3 mRNA expression in stromal cells, confluent stromal cells were
treated with steroid hormones in serum-free medium for 9 days and
cultured for an additional 24 h, as described in Materials
and Methods, in the absence or presence of increasing
concentrations of TGFß1 (0–40 ng/mL). Figure 7 shows a representative experiment with
dose-dependent up-regulation of both TIMP-1 (Fig. 7A) and TIMP-3 (Fig. 7B) mRNA expression with increasing concentrations of TGFß1. TGFß1
mediated up-regulation of TIMP-1 and TIMP-3 mRNA expression in stromal
cells was attenuated by anti-TGFß1 antibody (10 µg/mL; Fig. 8, A and B).
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The effects of ovarian steroids on PRL production by human endometrial stromal cells grown to confluence in standard medium and subsequently treated for 9 days with E and P in serum-free medium were determined. Under these conditions, there was no detectable level of PRL (<2 ng/106 cells) in conditioned medium from cells grown in the absence of steroid hormones (negative control). The PRL level in conditioned medium from cells grown in the presence of steroid hormones for 9 days was 4.26 ± 0.3 ng/106 cells (the mean ± SD of PRL levels obtained from 19 representative experiments). In addition, there was no detectable IL-1ß protein (<3.9 pg/106 cells) in conditioned medium from cultured human endometrial stromal cells grown in either the presence or the absence of steroids.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The stimulatory effect of IL-1 on 92-kDa type IV collagenase expression in cultured stromal cells is consistent with previous findings in human trophoblast (36), rat mucosal keratinocytes (55), synovial fibroblasts (56), and chondrocytes (57). It strongly supports the hypothesis that IL-1 may play a major role in the embryonic-maternal interaction at the level of the endometrial surface by degradation of extracellular matrix and tissue remodeling to initiate trophoblast invasion. TGFß, a putative regulator in the proliferation and differentiation of trophoblast cells (58), had no effect on 92-kDa type IV collagenase gene expression, but had a positive effect on both TIMP-1 and TIMP-3 mRNA expression. Both stimulated (36) and inhibitory (59) regulation of the 92-kDa type IV collagenase by TGFß have been described in first trimester trophoblast cultures. In the present study, endometrial stromal cells are nonresponsive to TGFß. However, the response of trophoblast tissue to TGFß indirectly enhances the IL-1-dependent increase in stromal cell 92-kDa type IV collagenase mRNA expression by further inhibiting the expression of TIMP-1 and TIMP-3.
The MMPs are a group of zinc enzymes responsible for matrix degradation of collagen and proteoglycans in the normal process of embryo implantation and tissue remodeling. Among MMP, 92-kDa type IV collagenase, also termed MMP-9, is expressed by implanting mouse embryos from days 5.5–7.5 (22). This enzyme has been implicated in human implantation, with first trimester trophoblast expressing uniquely high quantities of a gelatin-degrading protease whose activity could be attenuated by the natural inhibitors of metalloproteinase (60). Further study demonstrated that production and activation of the 92-kDa type IV collagenase are necessary for cytotrophoblast invasion in vitro (14). IL-1ß concentrations paralleled the invasive potential of human cytotrophoblasts, with the highest level produced by first trimester cells and the lowest levels produced by term cells (10).
The naturally occurring TIMP are likely to have an important physiological role in a wide variety of tissue-remodeling process, with TIMP-1 and TIMP-2 being the best studied. More recently, TIMP-3 has been isolated from chicken (61), mouse (62), and human (63). Both TIMP-1 and TIMP-3 have demonstrated inhibitory activity against the 72- and 92-kDa type IV collagenases (20, 64). TIMP-2 is closely related in action to TIMP-1 (19). Intensive expression of TIMP-3 in maternal cells surrounding invading embryonic tissue was demonstrated during mouse embryo implantation (22). The gene expression of TIMP-3 by human stromal cells also suggests that it may be important in the regulation of trophoblast invasion (65). In a recent report, the coincidental expression of the 92-kDa type IV collagenase and TIMP-1, TIMP-2, and TIMP-3 in the same decidual cells suggests that the regulation of 92-kDa type IV collagenase activity plays an important role in placental tissue organization and in the invasion of trophoblastic cells into the uterine wall (23).
We have found that stromal cells express IL-1R tI, suggesting that autocrine-paracrine stimulation of stromal cell TIMP-1, TIMP-3, and 92-kDa type IV collagenase gene expression by embryonic IL-1 (29) may be a critical receptor-mediated event, permitting the trophoblast to traverse the endometrial epithelium and initiate stromal cell implantation. Thus, we hypothesize that the vital process of blastocyst implantation may be dependent in part on a complex series of molecular and cellular events involving embryo-maternal communication via the IL-1 system. These cytokines are involved in the regulation of cell proliferation, differentiation, and programmed cell death (66). The immunohistochemical localization of the IL-1 system in early implantation sites provides additional evidence of the IL-1 system in trophoblast cells and decidual tissue (29, 67).
We have investigated the effects of IL-1ß and TGFß1 on their steady state levels of mRNA expression in cultured stromal cells using QC-PCR technology. RT-PCR can be used to analyze very low abundance mRNAs derived from cells or tissues and is now a well established technique whose sensitivity provides a major advantage. Quantitative analysis of these messages can be achieved by a modification known as QC-PCR (52, 68, 69, 70, 71), in which an internal control molecule possessing a small deletion in the amplified portion of the specific molecule is amplified simultaneously with the target sample instead of another control molecule, such as ß-Actin or globulin. In addition, because the efficiency of amplification of the internal control molecules is identical to that of the target template, quantitative PCR can avoid the discrepancies associated with tube to tube or sample to sample variations in the kinetics of the RT reaction (70). As quantitative PCR is based on the competitive status between the target molecules and internal standard molecules within the same PCR reaction, the relative amount of each product, expressed as a ratio of target molecule to internal standard molecule, is determined. When the ratio is between 0.66–1.5, the final result has an error of approximately 10%, and differences as small as 20% between two samples can be determined with an accuracy of 95% (54). In the present study, we constructed an internal standard with a defined deletion fragment from the target cDNA sequence. A defined amount of the internal standard cDNA determined by the equivalent range of target and internal standard cDNA was added to each PCR sample, allowing us to quantify the amount of specific target cDNA available. Thus, stromal cell 92-kDa type IV collagenase, TIMP-1, and TIMP-3 mRNA expressions are quantitatively regulated by IL-1 and TGFß. Our results provide indirect evidence that the IL-1 system may play a significant role as a molecular autocrine-paracrine regulator in embryo-endometrial interactions during implantation.
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Acknowledgments |
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Footnotes |
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Received October 21, 1997.
Revised January 21, 1998.
Accepted February 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and transforming
growth factor ß expression in endometrium, placenta, and placental
membrane. Am J Obstet Gynecol. 163:1430–1437.[Medline]
:
synergistic stimulation of metalloproteinases, PGE2 and
proliferation in human fibroblasts. Exp Cell Res. 210:166–171.[CrossRef][Medline]
and interleukin-1 ß proteins in
human placentas. J Reprod Immunol. 22:257–268.[CrossRef][Medline]
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J. S. Krussel, E. M. Casan, F. Raga, J. Hirchenhain, Y. Wen, H.-Y. Huang, P. Bielfeld, and M. L. Polan Expression of mRNA for vascular endothelial growth factor transmembraneous receptors Flt1 and KDR, and the soluble recetor sflt in cycling human endometrium Mol. Hum. Reprod., May 1, 1999; 5(5): 452 - 458. [Abstract] [Full Text] [PDF]
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F. Raga, E. M. Casañ, Y. Wen, H.-Y. Huang, F. Bonilla-Musoles, and M. L. Polan Independent Regulation of Matrix Metalloproteinase-9, Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), and TIMP-3 in Human Endometrial Stromal Cells by Gonadotropin-Releasing Hormone: Implications in Early Human Implantation J. Clin. Endocrinol. Metab., February 1, 1999; 84(2): 636 - 642. [Abstract] [Full Text]
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