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Original Studies |
Department of Gynecology and Obstetrics, Stanford University School of Medicine (H.W.C., Y.W., M.L.P.), Stanford, California 94305-5317; Department of Obstetrics and Gynecology, Ewha Womans University School of Medicine (H.W.C., J.J.A., H.S.M.), 158-710 Seoul, Korea
Address all correspondence and requests for reprints to: Dr. Hye-Won Chung, Department of Obstetrics and Gynecology, Ewha Womans University Mokdong Hospital, 911-1 Yang Chun Ku Mock 6 Dong 158-710 Seoul, Korea. E-mail: hyewon{at}mm.ewha.ac.kr
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Abstract |
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Introduction |
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The urokinase plasminogen activator (u-PA) system is associated with tissue remodeling, tumor invasion, ovulation, and embryo implantation (4, 5). The proenzyme plasminogen is transformed into the highly potent plasmin by activating enzymes, tissue plasminogen activator (t-PA), and u-PA. u-PA generates plasmin in events involving degradation of ECM and has been studied in relation to cancer metastasis (6). The u-PA receptor (u-PAR) has two types, u-PAR and soluble u-PAR (su-PAR), which arise by alternative splicing of the carboxyl-terminal end, suggesting a retained binding activity (7). The binding of u-PA to u-PAR is implicated in diverse biological processes such as cell migration, tissue remodeling, and tumor cell migration and invasion. u-PAR may enhance proteolytic activity on the cell surface, engender a mitogenic response, and favor endocytosis of complexed and free u-PA. Recent studies indicated that u-PAR can act as an ECM receptor during cell adhesion (8). su-PAR has retained u-PA-binding activity and is a water-soluble, secreted protein. The function of su-PAR is still unknown, but some reports suggest that su-PAR can increase the local availability and activity of u-PA by retarding its inhibition by PAI-1 and its clearance (9). The activity of u-PA is regulated by specific plasminogen activator inhibitors (PAIs). Type 1 PAI (PAI-1), a 50-kDa glycoprotein, is the major physiological inhibitor of both t-PA and u-PA and plays an important role in determining net fibrinolytic activity in vivo (10, 11, 12). Inappropriate expression of PAI-1 may suppress normal fibrinolytic activity of the tissues and result in pathological fibrin deposition (13, 14). In vitro decidualization of endometrial stromal cells is accompanied by diminished u-PA activity due to increasing decidual cell-derived PAI-1 both in vivo and in vitro (15). Up-regulation of u-PAR and elimination of the u-PA:PAI-1 complex via this receptor are greatly increased by progesterone.
Interleukin-1ß (IL-1ß) expression has been documented in human endometrium (16) and in the human periimplantation embryo (17) and is believed to play a role in embryo implantation (18). In addition, IL-1 affects PA activity in rat granulosa (19) and mesangial cells (20), induces a coordinated increase in u-PA and u-PAR in keratinocytes (21), and increases the production of u-PA and PAI-1 in colon carcinoma cells (22). Thus, we hypothesized that IL-1 might play a crucial role in embryo implantation by regulating stromal cell expression of u-PA, PAI-1, u-PAR, and su-PAR. Therefore, we examined the regulation of u-PA, PAI-1, u-PAR, and su-PAR messenger ribonucleic acid (mRNA) and protein expression in cultured human stromal cells by IL-1ß using quantitative competitive PCR and enzyme-linked immunosorbent assay (ELISA).
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Materials and Methods |
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Human luteal endometrial samples were obtained from surgical specimens of normally cycling women undergoing laparoscopic surgery or hysterectomy for various nonmalignant lesions. Patients with pelvic inflammatory disease and dysfunctional uterine bleeding were excluded. Written consent from the patients under a protocol approved by the institutional committee on the use of human subjects in research at Stanford University was obtained before collection of tissue samples for this study. Endometrial samples were taken using a curette in the operating room before laparoscopic procedures; in patients undergoing hysterectomy, the uterine cavity was opened, and endometrium was obtained immediately after the specimen was removed. The tissue samples used for this study were histologically normal. Stromal cells were separated from the glandular epithelium after collagenase dissection and were cultured using an established in vitro model, as previously described (23, 24), in 75% DMEM (Life Technologies, Inc., Grand Island, NY) and 25% MCDB-105 (Sigma, St. Louis, MO) containing antibiotics, 5 µg/mL insulin (Sigma), and 10% charcoal-treated FBS (Gimmini, Calabasas, CA). The homogeneity of cultures was determined by morphological characteristics and verified by immunocytochemical localization of vimetin as a marker for stromal cells. As defined by this method, endometrial stromal cell monolayers contained less than 1% endometrial epithelial or vascular cells (24).
Hormonal treatment
Stromal cells (passages 1–5) were plated at 2 x 105/well in 24-well culture plates (Falcon, Becton Dickinson and Co., Lincoln Park, NJ) and cultured in steroid-free 75% DMEM (Life Technologies, Inc.) and 25% MCDB-105 (Sigma) containing antibiotics, 5 µg/mL insulin (Sigma), and 10% charcoal-treated FBS (Gimmini). 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), 10 nmol/L estradiol (Sigma), 1 µmol/L progesterone (Sigma), 20 ng/mL epidermal growth factor (Sigma), and 1 mg/mL BSA (Irvine Scientific, Santa Ana, CA) for 9 consecutive days. Control confluent cells were cultured in the same medium without steroids, and medium was renewed every 2 days throughout the culture period. Conditioned serum-free medium was collected and frozen at -70 C until assayed for PRL, PAI-1, and u-PAR.
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/µg; Genzyme Corp., 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 Corp.) in a dose-dependent manner (1–20 µg/mL) for 24 h. Seven experiments using cells obtained from different patients were performed.
RNA analysis
Total RNA was extracted from cultured stromal cells using the guanidium isothiocyanate method (RNAzol, Tel-Test "B," Inc., Friendswood, TX). Total RNA was separated from DNA and proteins by adding chloroform and was precipitated using isopropanol. The precipitate was washed twice in 75% ethanol, air-dried, and rediluted in diethylpycocarbonate (DEPC)-treated dH2O. The amount and purity of extracted RNA were quantitated by spectrophotometry in a GenQuant RNA/DNA calculator (Pharmacia Biotech, Cambridge, UK). All experiments were performed using a minimum of seven samples from different women.
Primers for RT-PCR
Specific sequences of oligonucleotide primers for detecting
stromal cell expression of u-PA, u-PAR, su-PAR, and PAI-1 (7, 25, 26, 27) were obtained from the GenBank database of the National
Center for Biotechnology Information of the NIH (internet address:
http://www.2.ncbi.nlm.nih.gov/cgi-bin/GenBank). One corresponding set
of primers each for u-PA, u-PAR, su-PAR, and PAI-1 was found with the
help of the program OLIGO 5.0 Primer Analysis Software (National
Bioscience, Plymouth, MN) and synthesized by the protein, amino acid
and nucleic acid facility (Beckman Center, Stanford University,
Stanford, CA). The human ß-actin primers that were used to amplify an
external standard were obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). ß-Actin mRNA expression was employed as
an external positive control, being detected in all samples studied,
thus assuring the integrity of RNA extraction and the RT-PCR process.
The primer sequences, locations on the mRNA, and sizes of the amplified
fragments are listed in Table 1
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For RT-PCR, the GenAmp RNA PCR kit (Perkin-Elmer Corp., Foster City, CA) was used. Nineteen microliters of RT-Mastermix 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 (all from Perkin-Elmer Corp.), 100 IU Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), and 1 µg total RNA diluted in 1 µL DEPC-treated H2O and placed in a 0.5-mL thin wall PCR tube (Applied Scientific, South San Francisco, CA).
RT-Mastermix in PCR tubes was covered with 50 µL light white mineral oil (Sigma) and kept on ice until the RT. RT was carried out in the DNA Thermal Cycler 480 (Perkin-Elmer Corp.) using a program with the following parameters: 42 C for 15 min and 99 C for 5 min, then quenched at 4 C. After the reaction was completed, samples were stored at -20 C until the PCR. As a negative control, 1 µL cultured medium without RNA sample was subjected to the same RT reaction.
Construction of the competitive and target complementary DNA (cDNA) fragments for u-PA, PAI-1, u-PAR, and su-PAR
Seven hundred and fifty-three, 1458, 1234, and 913 bp of native
u-PA, PAI-1, u-PAR, and su-PAR (the target) were obtained by PCR
amplification of reverse transcribed total RNA from endometrial biopsy
with the regular 3'- and 5'-primers. The PCR product was visualized by
agarose gel electrophoresis stained with ethidium bromide, and the cDNA
was extracted from the gel, purified with an agarose gel extraction kit
(Pharmacia Biotech, Cambridge, UK), and quantitated by
spectrophotometry (Pharmacia Biotech). For the u-PA target
DNA, 702 bases were also sequenced and were found to have 99% homology
with the original u-PA sequence, thus confirming its identity. To
construct a competitive cDNA fragment, a floating primer with a
sequence complementary to the cDNA between the 3'- and 5'-primer
binding sites was designed by attaching the complementary sequence of
the binding site of the original 3' u-PA, PAI-1, u-PAR, and su-PAR
primer (Fig. 1). After PCR with the
regular 5'-primer and the 3'-floating primer, the PCR product was
visualized by agarose gel electrophoresis stained with ethidium
bromide. cDNA extraction, purification, and determination of the
concentration were performed as described above. This step resulted in
cDNA fragments of 462, 391, 296, and 128 bp deletion compared with the
target cDNA and with the 3'- and 5'-end primer binding sites on its
ends.
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The standard curve for u-PA, PAI-1, u-PAR, and su-PAR was
constructed by coamplification of a constant amount of competitive cDNA
(0.02, 0.1, 0.5, and 100 fmol, respectively) with declining amounts of
target cDNA (10–0.01,0.63–0.003, 16–0.032, and 624–0.61 fmol,
respectively) obtained by serial dilution. Sixty microliters of the
cDNA mix were added to 40 µL PCR-Mastermix containing 1.9 mmol/L
MgCl2 solution, 10 x PCR buffer II, 0.2
mmol/L of each deoxy-NTP, 2.5 U Taq polymerase (all from
Perkin-Elmer Corp.), corresponding paired primers in a
concentration of 0.2 µmol/L of each primer to a total volume of 100
µL, and 14.5 µL DEPC-treated H2O. The
reaction was covered with 50 µL light white mineral oil and put in
the Perkin-Elmer Corp. DNA Thermal Cycler 480. PCR cycles
were started at 95 C for 5 min to denature all proteins; 30 cycles for
45 s at 94 C; 45 s at 70 C for u-PA, u-PAR, and su-PAR;
45 s at 55 C for PAI-1; and 60 s at 72 C. The reaction was
terminated at 72 C for 5 min and was quenched at 4 C. Two percent
agarose gel (Life Technologies, Inc.) 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, Inc.) 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, Inc., Hercules, CA). The
logarithmically transformed ratios of target cDNA to competitive cDNA
were plotted against the log amount of initially added target cDNA in
each PCR to obtain the standard curve shown in Fig. 2. This standard curve was highly
reproducible and linear. The values obtained from the regression line
of the standard curve (y = b +
mx) allowed us to calculate the amount of cDNA transcripts
in an unknown sample: 0.02 fmol u-PA, 0.1 fmol PAI-1, 0.5 fmol u-PAR,
and 100 fmol su-PAR competitive cDNA were added to each unknown sample
before PCR. The ratios of the densities of sample target cDNA band
(753, 1458, 1234, and 913 bp) to competitive cDNA (291, 1067, 938, and
785 bp) were logarithmically transformed and compared with the values
obtained from standard curve. Quantitative competitive PCR was carried
out on at least two aliquots from the reverse transcriptase cDNA of
each patient, and the results did not differ more than ±5%.
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PAI-1 and u-PAR in conditioned medium derived from the end of cultures was measured by ELISA (American Diagnostica, Inc., Greenwich, CT) that detects PAI-1 and both u-PAR and su-PAR with a detection limit of 50 pg/mL and intra- and interassay coefficients of variation less than 7% and 8.8%, respectively. All samples were assayed in duplicate. As a marker of decidualization, PRL in conditioned medium was measured with an ELISA kit (Diagnostics Systems Laboratories, Inc., 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.
Data analysis
Statistical analysis was performed using ANOVA and a post-hoc test with a t test. The statistical analysis was carried out using Statistical Package for Social Science Package version 9.0 (SPSS, Inc., Chicago, IL), with P < 0.05 considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To confirm the expression of u-PA, PAI-1, u-PAR, and su-PAR mRNA in cultured endometrial stromal cells, confluent stromal cells were treated with estrogen and progesterone in serum-free medium for 9 days and then cultured for an additional 24 h in the absence or presence of rhIL-1ß (100 IU/mL). Total endometrium, rhIL-1ß-treated stromal cells and nontreated stromal cells all expressed u-PA, PAI-1, u-PAR, and su-PAR (data are not shown).
Dose-response study of IL-1ß-mediated regulation of stromal cell u-PA, PAI-1, u-PAR, and su-PAR mRNA expression
To further assess the IL-1ß-mediated regulation of u-PA, PAI-1,
u-PAR, and su-PAR 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 in the absence or
presence of increasing concentrations of rhIL-1ß (0–1000 IU/mL).
Figure 3 shows target cDNA concentration
documenting a dose-dependent up-regulation of PAI-1, u-PAR, and su-PAR
at IL-1ß concentrations of 1,000, 1,000, and 10 IU/mL, respectively
(P < 0.05). u-PA mRNA expression did not appear to be
dose dependent on IL-1ß concentration.
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The confluent stromal cells were treated with steroid hormones in
serum-free medium for 9 days and cultured for an additional 24 h
in the rhIL-1ß (100 IU/mL) plus increasing concentration of
anti-IL-1ß antibody (Fig. 4, A–D). The
increase in PAI-1, u-PAR, and su-PAR mRNA expression in the presence of
rhIL-1ß (100 IU/mL) was attenuated with increasing concentrations of
anti-IL-1ß antibody (P < 0.05). The u-PA mRNA
expression was not influenced by anti-IL-1ß antibody.
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PAI-1 and combined u-PAR and su-PAR protein levels in
conditioned medium from cultured stromal cells increased as a
dose-dependent function of IL-1ß concentrations as measured by ELISA.
PAI-1 was significantly increased in the presence of 1000 IU IL-1ß
(Fig. 5A), and this increase was
attenuated with increasing concentrations of anti-IL-1ß antibody
(Fig. 6A; P < 0.05).
Combined u-PAR and su-PAR was significantly increased in the presence
of 100 IU IL-1ß (Fig. 5B), and this increase was attenuated with
increasing concentration of anti-IL-1ß antibody (Fig. 6B;
P < 0.05).
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The effect of ovarian steroids on PRL production by human endometrial stromal cells grown to confluence in serum-free medium was 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 10 nmol/L estradiol (Sigma) and 1 µmol//L progesterone (Sigma) for 9 days was 4.25 ± 0.3 ng/mL (the mean ± SD of PRL levels obtained from 25 representative experiments).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The plasmin/PA cascade is an essential component in the tissue remodeling that accompanies repair processes. PA converts plasminogen to plasmin, a potent serine proteinase that solubilizes fibrin, digests ECM proteins, activates procollagenase, and degrades various basement membrane components. Human trophoblast cells secrete u-PA (28) and display saturated cell surface receptors (29). Endometrial stromal cell cultures secrete both u-PA and t-PA, with progesterone lowering the secretion of u-PA (16). There are two members of the serine family of inhibitors, the endothelial-type inhibitor PAI-1 and the placental-type inhibitor PAI-2, which bind to both PAs with high affinity and inactivate them (14). Stabilization of PAI-1 in its active form, by binding to circulating vitronectin (14) and to ECM-sequestered vitronectin (30), is integral to the regulation of fibrinolysis and proteolysis of ECM components, respectively. PAI-1, like u-PA, is under the control of progesterone. Progesterone increased the stability of PAI-1 mRNA in endometrial stromal cells (31) and its release into conditioned medium, whereas u-PA decreased such release (32). Cultured stromal cells secrete more PAI-1 than PAI-2, and this preferential production of PAI-1 during in vitro decidualization has important implications when extrapolated to decidual cell involvement in regulating periimplantational trophoblast invasion. Such decidual cell-derived PAI-1 could act as a barrier to invasion of the endometrial ECM by trophoblastic cells.
u-PA binds to its cell surface receptor (u-PAR) and is implicated in diverse biological processes such as cell migration, tissue remodeling, and tumor cell invasion. Recent studies indicated that u-PAR can act as an ECM receptor during cell adhesion (8). u-PAR has two spliced variants, u-PAR and su-PAR. su-PAR is a soluble form, which is encoded by the same gene as the surface receptor. The binding domain for u-PA is similar, but the carboxyl-terminal end by which the surface receptor is anchored to the cell membrane is modified through alternative splicing, which suggests a retained binding activity (7). Along with u-PA, its cell surface receptor (u-PAR) is also believed to be involved in recruiting u-PA within the tumor cell environment. Overexpression of u-PAR increased invasive capacity 4- to 5-fold, demonstrating the role of u-PAR in tumor progression (33). The u-PA/u-PAR complex remains available for efficient inhibition by PAIs, which may therefore play a major role in controlling cell surface plasminogen activation and extracellular proteolytic activity. u-PAR expression was seen in the trophoblast, decidual cells, and endometrial tissues of ectopic pregnancy (34); ovarian follicles (35); and cultured stromal cells (17).
Human endometrium is an active site for cytokine production and action (36, 37). IL-1 may play a critical role as an autocrine/paracrine cytokine and major local regulator of steroid hormone action (38), implicating it in the implantation process (39). The presence of a complete IL-1 system, including IL-1ß mRNA expression (40), IL-1 receptor type I (41), and IL-1 receptor antagonist (42), has been documented in human endometrium. There is immunoreactive evidence of the IL-1 system in the materno-trophoblast unit (40). The IL-1 concentration paralleled the invasive potential of human cytotrophoblast, with the highest level produced by first trimester cells, and the lowest levels produced by term placenta (43). Stromal cells express IL-1 receptor type I (40), suggesting that autocrine-paracrine stimulation of stromal cell PAI-1, u-PAR, and su-PAR gene expression by embryonic IL-1 may be an important receptor-mediated event, permitting the trophoblast to initiate stromal cell implantation.
Due to its extraordinarily high sensitivity, PCR is widely used for amplifying cDNA copies of low abundance mRNA (44, 45). However, quantitation is unreliable because the amount of PCR product increases exponentially with each cycle of amplification; therefore, minute differences in any of the variables that affect the efficiency of amplification can dramatically alter product yield. Coamplification with a different reporter gene product (46, 47) is a semiquantitative mRNA quantitation method. Thus, we constructed an internal standard with a defined deletion fragment from the target cDNA and used the same primers to coamplify the unknown and the competitor, allowing us to quantify the amount of specific target cDNA available. 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 (48). For RNA quantitation, Northern blots are widely used. However, the Northern blot technique requires at least 10 µg total RNA for semiquantitation, whereas QC-PCR requires only 1 µg total RNA and is useful when only small amounts of tissue, such as endometrial currettings, are examined.
Thus, we have investigated mRNA expression in small samples of cultured human endometrium cells using QC-PCR technology and quantified the amount of specific target u-PA cDNA. Stromal cell u-PAR, su-PAR, and PAI-1 mRNA expression is quantitatively increased by IL-1ß. Our results provide indirect evidence that the IL-1 system may play a significant role as a molecular autocrine-paracrine regulator in embryo-endometrial interaction during implantation. Successful implantation may require a substantial local IL-1ß concentration to support PA system proteolysis and favor trophoblast invasion.
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Acknowledgments |
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Footnotes |
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Received November 5, 1999.
Revised June 2, 2000.
Revised November 6, 2000.
Accepted November 9, 2000.
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References |
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, and transforming
growth factor-ß expression in endometrium, placenta, and placental
membranes. Am J Obstet Gynecol. 163:1407–1430.
. Exp Cell Res. 223:395–404.[CrossRef][Medline]
inhibits in-vitro decidualization of human
endometrial stromal cells. Hum Reprod. 9:2411–2447.This article has been cited by other articles:
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Y. H. Ng, H. Zhu, C. J. Pallen, P. C.K. Leung, and C. D. MacCalman Differential effects of interleukin-1beta and transforming growth factor-beta1 on the expression of the inflammation-associated protein, ADAMTS-1, in human decidual stromal cells in vitro Hum. Reprod., August 1, 2006; 21(8): 1990 - 1999. [Abstract] [Full Text] [PDF]
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