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The Impact of the Human Genome on Endocrinology: Original Articles |
Center for Reproductive Science, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, California 94143-0556
Address all correspondence and requests for reprints to: Robert N. Taylor, M.D., Ph.D., Center for Reproductive Science, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, HSE 1689, Box 0556, San Francisco, California 94143-0556. E-mail: . rtaylor{at}socrates.ucsf.edu
Abstract
RANTES (regulated on activation, normal T cell expressed and secreted) is synthesized by endometrial and endometriotic stromal cells and circulates in peritoneal fluid. Reports indicate that medroxyprogesterone acetate (MPA) is clinically effective in alleviating pelvic pain in the majority of endometriosis patients, which leads us to hypothesize that MPA may be antiinflammatory.
Prolonged treatment (8 d) with MPA resulted in 36% and 50% decreases in luciferase activity and RANTES protein production, respectively, whereas shorter treatment (2 or 4 d) with MPA had no significant effect. We also observed that 8 d of MPA increased PR expression. Both effects were blocked by RU486. Cotransfection of endometrial stromal cells with PR enhanced the effects mediated by endogenous PR. In addition, its action via progesterone response element cis-elements, PR appeared to inhibit trans-activation of a nuclear factor-
B-responsive element, further suppressing RANTES expression.
These studies indicate that prolonged progestin exposure down-regulates endometrial RANTES gene transcription in vitro. The effect is PR dependent and mediated in part through a nuclear factor-B pathway. The clinical effectiveness of chronic progestin treatment in endometriosis-associated pelvic pain may be attributed to its inhibition of RANTES production and its suppression of inflammatory responses in the pelvis.
ENDOMETRIOSIS IS a complex gynecological disorder associated with pelvic pain and infertility that affects between 5–15% of women of reproductive age (1). Although no single theory can fully account for the diverse clinical presentations of endometriosis, Sampson’s hypothesis (2) of retrograde menstruation is most consistent with the gravitationally dependent location of implants on the peritoneal surfaces of the ovaries, broad ligaments, and cul-de-sac. Retrograde menstruation is an almost universal phenomenon (3), and with each cycle, some endometrial tissue is shed into the peritoneal cavity. The expression of adhesion molecules, angiogenic proteins, or chemotactic factors may account for the pathological sequelae of endometrial implants in the peritoneal cavity. Immune cells and their secretory products have been recognized as important mediators of the pathophysiology of endometriosis (4, 5). Cytokines liberated by these cells (e.g. IL-1ß and TNF
) appear to play a role in the pathways associated with pain perception (6, 7). However, no clear correlation between peritoneal fluid concentrations of IL-ß and TNF and pelvic pain was observed in a clinical study of endometriosis (8). Medroxyprogesterone acetate (MPA) is clinically effective in reducing pain and improving overall well-being in the majority women with endometriosis (9, 10). Further, this relatively inexpensive therapy is as effective as other medical treatments, such as GnRH analogs and danazol. It has been proposed that symptom resolution with these latter agents results from pituitary gonadotropin inhibition and regression of the endometriotic implants secondary to ovarian hormone suppression (11).
Our laboratory has characterized the expression and regulation of a specific chemokine, RANTES (regulated on activation, normal T cell expressed and secreted). RANTES is an 8-kDa chemoattractant for monocytes and activated T cells (12), the two predominant leukocytes in peritoneal fluid of women with endometriosis (13). RANTES is one of the key chemokines expressed by normal endometrial and endometriotic stromal cells (14), and its concentration (15) and bioactivity (16) are elevated in peritoneal fluid of women with endometriosis. The objective of the current study was to investigate whether MPA and other synthetic progestins could inhibit RANTES gene expression in isolated endometrial stromal cells in vitro.
Subjects and Methods
Patient recruitment, tissue acquisition, and characterization
Healthy women with ovulatory menstrual cycles, who had not received hormones or GnRH agonist therapy for at least 6 months before surgery, were enrolled in this study. Subjects undergoing elective surgery for leiomyomata uteri or other benign uterine conditions were recruited for normal endometrial biopsies. Biopsies were obtained after the patients provided written informed consent, under a study protocol approved by the committee on human research at University of California-San Francisco. All samples were obtained in the proliferative phase of the cycle, and this was confirmed histologically according to criteria of Noyes et al. (17).
Human endometrial stromal cell cultures
Stromal cells were separated from glandular cells and debris by filtration through narrow gauge sieves. Stromal cells were plated and subcultured to eliminate contamination by macrophages or other leukocytes. Extensive characterization of cell cultures prepared using this protocol confirmed that they were more than 95% pure and retained functional markers of their endometrial origin (18).
Treatment of stromal cells with hormones and antihormones
Steroid hormones and vehicle controls were added in 0.1% ethanol carrier directly to the cells growing in 10 ml MEM with 10% FCS (HyClone Laboratories, Inc., Logan, UT) in T75 flasks. The concentrations and sources of ligands are listed as follows: MPA (10-7 M; Sigma, St Louis, MO), and the progestin agonist promegestone (R5020, 17,21-dimethyl-19-norpregna-4,9-dien-3,20-dione) 10-7 M) and the antagonist mifepristone [RU486, 17ß-hydroxy-11ß-(4-dimethylamino-phenyl)-17-(1-propynyl)-estra-4,9-dien-3-one; 10-7 M; gifts from Dr. D. Philibert, Roussel-UCLAF, Romainville, France]. Media were changed every 2 d, with replenishment of the steroid ligands at each change, for up to a total of 8 d of treatment. Prior experiments had determined this progestin concentration to maximally induce PRL (19) and glycodelin (20), and it is achievable in the circulation of women receiving MPA clinically (21, 22). Similar results were observed in phenol red-containing and phenol red-free medium.
Analysis of PR, nuclear factor-B (NF-B), and inhibitor of -B (I-B) proteins in endometrial stromal cell lysates
Media were removed by aspiration, and the cells were washed with 10 ml cold Tris-buffered saline and scraped into 500 µl extraction buffer [20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1 mM Pefablock (Pentapharm AG, Basel, Switzerland), 2 µM leupeptin, 0.14 U/ml aprotinin, and 1 mM vanadate]. Cells were burst by repeated freeze/thaw cycles and were centrifuged at 12,000 rpm for 15 min at 4 C. Supernatants of extracts were denatured in sample buffer, and 100 µg total protein were electrophoresed on 8% polyacrylamide SDS-gels at 30 mA for 2 h. Proteins were transferred to Immobilon polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) for 2 h in a semidry transfer system (Hoefer Scientific, San Francisco, CA). Membranes were blocked for 1 h in Tris-buffered saline-Tween plus 5% instant nonfat dry milk, then probed with 10 µg/ml of the anti-PR monoclonal antibody (AB52), NF-B p65 (SC109, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or I-B (C-terminal peptide that detects both phosphorylated and non phosphorylated protein, gift from Dr. W. Greene, Gladstone Institute, University of California-San Francisco) at 4 C overnight. T47D breast cancer cells were used as a positive control for PR and were cultured on plastic wells for 2 d in complete medium. The polyvinylidene difluoride membranes were washed in the same solution before the addition of a 1:5000 dilution of horseradish peroxidase-conjugated antimouse IgG, and specific bands were visualized by the ECL (Amersham Pharmacia Biotech, Arlington Heights, IL) method on Kodak X-OMAT film (Eastman Kodak Co., Rochester, NY). Differences in Western blots were estimated by transmission scanning using NIH Image 1.60 software. All experiments were repeated to assure reproducibility. Membranes were stripped and reblotted with ß-actin (SC8432, Santa Cruz Biotechnology, Inc.) as an internal control for protein loading and transfer.
RANTES ELISA
A specific sandwich ELISA was used to quantify RANTES in conditioned media (Quantikine, R\|[amp ]\|D Systems, Inc., Minneapolis, MN). In our laboratory the assay was linear and sensitive to 8 pg/ml, with intra- and interassay coefficients of variation of 3.2% and 6.5%, respectively. The assay is specific for human RANTES, with no known cross-reactivity with other cytokines or chemokines (R\|[amp ]\|D Systems, Inc., 2001 Immunoassay catalog). Aliquots of culture supernatants were each tested in duplicate at several dilutions and compared with reference standards of recombinant human RANTES (R\|[amp ]\|D Systems, Inc.).
Reporter gene and expression vectors
A 477-bp human RANTES promoter-luciferase fusion construct (-456/+20) was subcloned into the pGL2 vector (Promega Corp., Madison, WI). We previously determined that this construct contained the same trans-activating function in endometrial stromal cells as the full-length promoter (23). A 236-bp promoter construct (-215/+20) was made by truncating the -456/+20 plasmid. The NF-B element of the wild-type RANTES promoter GGGGATGCCC (at nucleotide -40/-31 was mutated to GTGTATGCCC with QuickChange site-directed mutagenesis kits (Stratagene, La Jolla, CA) using oligonucleotides containing the desired mutation. The NF-B response element reporter construct was a gift from Dr. Dale Leitman (University of California-San Francisco). Dr. Anne Mantel-Guiochon (Le Kremlin-Bicetre, France) provided plasmids expressing human PR-B (pGS5-hPR), and those expressing human PR-A were gifts from Dr. Geoff Greene (University of Chicago, Chicago, IL). None of the constructs or mutants contained heterologous promoter sequences, and they were sequenced by the University of California-San Francisco Biomolecular Resource Center to verify that the correct sequences and mutations were present.
Cell transfections and luciferase assay
Transient transfections were performed in human endometrial stromal cells grown in MEM with 10% FCS and antibiotics in 12-well plates at about 50% confluence. pGL2-RANTES promoter (0.45 µg; firefly luciferase, experimental reporter) was added to each well using Effectene reagents (QIAGEN, Chatsworth, CA). The RANTES promoter transfection efficiencies were normalized to an independent control plasmid (0.1 µg Renilla luciferase reporter), cotransfected simultaneously. The cells were then incubated for 24 h in MEM with 10% FCS and antibiotics before luciferase activity was quantified. The results are presented as the percentage of luciferase activity between treated cells and untreated cells after correcting for transfection efficiency. Each reporter vector was assayed in at least three independent cultures. Empty pGL2 vector was analyzed as a control and revealed low basal activity.
Data presentation and statistical analysis
RANTES promoter luciferase assay data are expressed as the mean ± SD for at least three independent experiments. The number of experiments is represented as n in Results. As normalized data were expressed as a percentage of the controls, the results were compared with nonparametric Mann-Whitney or Kruskal-Wallis tests using StatView software. Two-tailed tests with P less than 0.05 were considered significant.
Results
Western analysis of PR in endometrial stromal cells
We previously demonstrated that primary cultures of endometrial stromal cells express functional PR (24). However, the effects of chronic treatment with MPA and RU486 have not been reported. Western blot analysis of samples from isolated endometrial stromal cells revealed two bands corresponding to the 116-kDa PR-B and 81-kDa PR-A isoforms (Fig. 1A
), identical to the PR isoforms reported by Tseng et al. (25) in these cells. We verified that the predominant PR isoform expressed in normal endometrial cells was PR-B (Fig. 1A). Incubation with MPA for 8 d significantly increased the content of PR-B in stromal cells by 50%, but only had a small effect on PR-A. Shorter durations of MPA exposure (2 and 4 d) had no significant effect on PR expression (Fig. 1A). This effect is probably mediated through the PR per se, as an equimolar concentration of RU486 partially inhibited the effect of MPA on PR up-regulation (Fig. 1B). The concentration of PR proteins in endometrial stromal cells was only 10% of that detected in T47D breast carcinoma cells, selected for their high levels of this protein.
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As we reported previously, untreated endometrial stromal cells have undetectable RANTES protein secretion, but this can be induced by treatment with TNF (14). However, 8-d MPA treatment inhibited the secretion of immunoreactive RANTES protein from stromal cells stimulated with 10 ng/ml TNF by 50 ± 1% (P < 0.05).
Effects of MPA on transcriptional inhibition of the RANTES promoter
To assess the roles of specific domains within the RANTES promoter, endometrial stromal cells were transiently transfected with wild-type RANTES promoter constructs (-456/+20 bp) cloned upstream of the firefly luciferase reporter gene. Short-term (2-d) MPA exposure had no effect on RANTES transcriptional activity (Fig. 2). However, in cells that had been subjected to 8 d of MPA, a 36 ± 17% (n = 10; P < 0.05) decrease in RANTES promoter transcription was observed (Fig. 2). RU486 blocked the MPA-induced inhibition of RANTES promoter activity (Fig. 2). The empty pGL2 vector background luciferase activity is less than 10% of the control -456/+20 RANTES reporter. A marked inhibitory effect of long-term progestins (79 ± 12%; n = 4; P < 0.05) was observed when PR-B expression vectors were cotransfected into stromal cells (Fig. 3). In the absence of progestin ligand, PR-B transfection had no effect on RANTES promoter activity. Progestin plus PR-A transfection did not significantly suppress RANTES promoter trans-activation (data not shown).
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Site-directed mutagenesis (Fig. 3
) of an NF-B site in the -215/+20 bp RANTES promoter inhibited luciferase activation relative to the -456/+20 construct (91 ± 2%; n = 4; P < 0.05); however, there was no further inhibition by progestin treatment with PR-B overexpression (Fig. 3). The empty pGL2 vector had low intrinsic activity.
Effects of MPA on NF-B protein and transcriptional inhibition of the NF-B response element
The data reported in Fig. 3 and previous studies from our laboratory indicated that NF-B signaling was critical to cytokine-induced RANTES promoter activation (23). We tested the hypothesis that 8 d of MPA might down-regulate steady state concentrations of NF-B protein in the stromal cells. Western blotting demonstrated that NF-B p65 was decreased by MPA, and this effect was reversed by RU486 (Fig. 4). I-B protein, the endogenous inhibitor of NF-B, was not affected by MPA (data not shown).
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B signaling, cells were transfected with PR-B and an NF-B response element construct. Treatment with MPA or R5020 markedly inhibited NF-B signaling by 92 ± 6% (n = 4; P < 0.05; Fig. 5).
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Pelvic pain and infertility, the primary symptoms of endometriosis, are believed to be caused by inflammatory responses initiated within endometriotic lesions (4, 5). Chronic progestin treatment is clinically effective in improving pain and somatic symptoms in the majority of women with endometriosis (1, 2), but does not benefit infertility (9). The mechanism of MPA and other progestins in this regard remains to be explained. It has been argued that their efficacy, like that of danazol, a synthetic androgen, and GnRH analogs, results from pituitary hormone inhibition and shrinkage of endometriotic lesions (11). Another possible hypothesis is that MPA alleviates inflammation at the sites of endometriotic implants. Our work has focused on RANTES, a critical chemokine in the pathogenesis of endometriosis (14, 15, 16) and one that we postulate plays an early role in the inflammatory response.
The current experiments were performed predominantly with normal endometrial stromal cells due to easier access and the results of previous work showing that normal and ectopic endometrial stromal cells were similar with respect to RANTES gene expression (14). Also, we observed identical RANTES promoter responses in three independent preparations of endometriotic stromal cells. Although endometrial and endometriotic epithelial cells do not express RANTES, paracrine factors derived from epithelia play a role in stromal RANTES gene regulation. We currently are examining this hypothesis in our laboratory.
The effect of MPA on stromal cells was first evaluated using Western analysis of PR proteins. Although short-term treatment of MPA (2 and 4 d) had no effect on PR, long-term exposure to MPA (8 d) significantly increased the amount of PR-B isoforms. Others made similar observations previously (25). The up-regulation of PR was inhibited by RU486, suggesting that the response is directly PR-induced. Although RU486 is reported to have antiglucocorticoid activity (26, 27, 28), we interpret its antagonism to be PR mediated. Cortisol (10-8 M) had no effect on RANTES promoter-reporter activity in our cell system (data not shown).
To investigate whether MPA could down-regulate RANTES gene expression, we established a model system of transiently transfected human RANTES promoter-luciferase reporter constructs in primary human endometrial stromal cells. The results showed that a short treatment with MPA had no effect, whereas 8-d exposure to MPA resulted in a 36% decrease in luciferase activity. This inhibitory effect was increased to 80% when PR-B was overexpressed in the stromal cells. RU486 blocked MPA-induced RANTES promoter down-regulation. These results support the hypothesis that MPA inhibits RANTES gene expression via a PR-mediated suppression of gene transcription.
Examination of the human RANTES promoter sequence reveals two consensus PRE at -284/-278 and -260/-254 bp (Fig. 3). Truncation of the promoter to remove these sites resulted in a loss of only about 40% of the inhibitory activity of PR-B-ligand complexes. This finding indicated that regions other than the consensus PRE play an important role in PR-mediated RANTES gene inhibition. PR-hormone complexes also have been shown to alter gene transcription through other mechanisms (29, 30, 31), and progesterone can inhibit RANTES production in lymphocytes via a PR-independent mechanism (32).
NF-B is a widely expressed, inducible transcription factor of particular importance to cells of the immune system (33). Previous studies from our laboratory proved that cytokines (e.g. IL-1ß) positively regulate RANTES gene expression via the NF-B pathway (23). In the current study we show that chronic MPA inhibition of NF-B transcription activity is probably PR dependent, with PR-B mediating a more potent repression than PR-A. I-B protein was not up-regulated by MPA treatment, which suggested that the down-regulation of NF-B by MPA treatment may not be through the accumulation of I-B.
We also observed that RANTES gene expression fell significantly when one of the NF-B cis-acting response element sites was mutated. We postulate that the inhibition of RANTES transcription by progestins is a PR-mediated effect involving sequestration of NF-B, as has been described in other systems (31).
The clinical utility of chronic MPA treatment in patients with endometriosis is still under debate. Our present study suggests that long-term MPA therapy could have beneficial effects on pelvic pain by suppressing the inflammatory response within endometrial implants. The effect is PR dependent, and the PR-B isoform appears most effective in down-regulating RANTES transcription. However, it is reported that PR-B is decreased or absent in endometriotic tissue (34), and this may account for the poor response to chronic MPA therapy in some women with endometriosis.
Strategies that ensure induction of functional PR-B within ectopic endometrium combined with long-term PR agonists such as MPA may offer promising future therapies for endometriosis.
Acknowledgments
We thank Victor A. Chao for his expert assistance with plasmid preparation, site-directed mutagenesis, and sequence verification.
Footnotes
This work was supported by a grant from the NICHHD, NIH, through Cooperative Agreement U54-HD-37321, as a part of the Special Cooperative Centers Program in Reproduction Research and by a fellowship from Pharmacia Corp.
Abbreviations: I-B, Inhibitor of -B; MPA, medroxyprogesterone acetate; NF-B, nuclear factor-B; PRE, progesterone response element; RANTES, regulated on activation, normal T cell expressed and secreted.
Received July 5, 2001.
Accepted January 28, 2002.
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