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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0972
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 12 6441-6447
Copyright © 2005 by The Endocrine Society

Sulindac Suppresses Nuclear Factor-{kappa}B Activation and RANTES Gene and Protein Expression in Endometrial Stromal Cells from Women with Endometriosis

Fritz Wieser, Jean-Louis Vigne, Isabelle Ryan, Daniela Hornung, Schima Djalali and Robert N. Taylor

Division of Gynecological Endocrinology and Reproductive Medicine (F.W., S.D.), University of Vienna, A-1090 Vienna, Austria; Department of Obstetrics and Gynecology (F.W., J.-L.V., I.R., R.N.T.), Center for Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143-0556; and Department of Obstetrics and Gynecology (D.H.), University of Schleswig-Holstein, 23538 Luebeck, Germany

Address all correspondence and requests for reprints to: Robert N. Taylor, M.D., Ph.D., Department of Gyn/Ob, Emory University School of Medicine, Woodruff Memorial Building, 1639 Pierce Drive, Room 4217, Atlanta, Georgia 30322. E-mail: robert.n.taylor{at}emory.edu.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: The nuclear factor-{kappa}B (NF-{kappa}B) pathway is a critical mediator of RANTES (regulated on activation, normal T cell expressed and secreted) gene regulation and therefore represents a potential target for therapy of endometriosis-associated symptoms.

Objective: The objective of this study was to investigate the effects of the antiinflammatory drug sulindac on NF-{kappa}B activation, NF-{kappa}B-mediated gene expression, RANTES gene and protein expression in endometrial stromal cells isolated from women with endometriosis, and unaffected controls.

Design: This was a clinical experimental study.

Setting: The study was conducted at a university hospital.

Results: The inflammatory response in endometriosis is augmented by a 5-fold increased TNF{alpha}-induced RANTES secretion from ectopic endometriotic stromal cells, compared with normal endometrial stromal cells (P < 0.05). Western blot analysis revealed basal activation of NF-{kappa}B in endometriotic cells, which could be suppressed by sulindac. EMSAs showed that sulindac dramatically decreased NF-{kappa}B activation and diminished TNF{alpha} and IL-1ß-induced NF-{kappa}B DNA binding activity. Sulindac pretreatment resulted in a significant decrease in TNF{alpha}-induced luciferase activity of NF-{kappa}B response element and –477 bp RANTES promoter constructs in normal and endometriotic stromal cells. The addition of sulindac to IL-1ß- and TNF{alpha}-treated endometriotic stromal cells also resulted in a 4-fold inhibition of RANTES protein secretion (P < 0.05).

Conclusions: We have demonstrated that sulindac exerts strong antiinflammatory effects by suppression of NF-{kappa}B translocation, inhibition of NF-{kappa}B-mediated gene transcription, RANTES gene expression, and protein secretion in normal and endometriotic stromal cells. These results suggest that drugs targeting the NF-{kappa}B pathway may be beneficial in the treatment of endometriosis-associated symptoms.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
ENDOMETRIOSIS IS DEFINED by the presence of endometrial cells outside the uterine cavity, which appear to arise due to retrograde menstrual dissemination (1). The disease affects about 10% of all reproductive-aged women and the prevalence rises to 20–50% in infertile women (2). The implantation hypothesis is the most widely accepted (1), supported by observations that retrograde menstruation and intraperitoneal spillage of viable endometrial cells occur frequently in cycling women and more commonly in those with genital outflow tract obstruction (3, 4, 5).

Increased production of chemokines such as RANTES (regulated on activation, normal T cell expressed and secreted) and monocyte chemoattractant protein (MCP)-1 in endometriosis tissues is postulated to promote a proinflammatory environment that fuels its pathogenesis (6, 7, 8, 9, 10, 11). RANTES is produced by endometriotic stromal cells and peritoneal macrophages themselves (7, 8, 9). Its levels in the pelvic fluid correlate with the severity of disease (6) and contribute to the majority of monocyte chemoattractant activity observed in vitro and in murine assays (7, 12). The nuclear factor-{kappa}B (NF-{kappa}B) pathway is involved in the induction of RANTES gene expression (8) and other genes including TNF{alpha}, IL-1ß, IL-6, IL-8, and MCP-1 in normal endometrial and endometriotic stromal cells (13, 14, 15, 16). Progesterone, a physiological NF-{kappa}B inhibitor, and other synthetic progestins, e.g. medroxyprogesterone acetate, commonly used in the clinical management of endometriosis (17) can suppress the NF-{kappa}B pathway (18, 19). We previously showed that the inhibitory effect of medroxyprogesterone acetate on RANTES expression in endometrial stromal cells is partly due to suppression of NF-{kappa}B action (20).

The NF-{kappa}B pathway also can be targeted by nonhormonal therapeutic agents such as the nonsteroidal antiinflammatory drugs (NSAIDs) including aspirin, diclofenac, naproxen, celecoxib, rofecoxib, and sulindac (21). NSAIDs with anti-NF-{kappa}B activity, including sulindac, have been shown to exhibit antiinflammatory effects by decreasing cyclooxygenase activity (21) and antiproliferative properties in cancer cells by decreasing cyclin D expression (21, 22). Among several NSAIDs we have screened, sulindac was described as one of the strongest NF-{kappa}B inhibitors (23). Sulindac is a nonselective cyclooxygenase (COX) inhibitor. It has been noted to have less vasoactive side effects on fetal renal and ductus function than indomethacin (24).

In the current study, we examined inducible RANTES secretion in normal endometrial and endometriotic stromal cells. We next evaluated the effect of sulindac on NF-{kappa}B-mediated gene transcription and NF-{kappa}B activation, RANTES gene expression, and protein secretion in normal endometrial and endometriotic stromal cells as a proof of concept for this class of potential pharmaceutical agents. Based on our findings in vitro, we suggest that sulindac might also be efficacious for treatment of endometriosis-associated symptoms.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Reagents

TNF{alpha} and IL-1ß and were purchased from R&D Systems (Minneapolis, MN). Sulindac was obtained from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Hyclone (Salt Lake City, UT). The mouse anti-NF-{kappa}B, p65 subunit monoclonal antibody was purchased by Chemicon International (Temecula, CA).

Patient recruitment and characterization

Reproductive-aged women (21–40 yr old) with regular menstrual cycles who had not received hormones or GnRH agonist therapy for at least 6 months before surgery were recruited after they had provided written informed consent under a protocol approved by the Committee on Human Research at the University of California, San Francisco. At laparoscopy, 10 women who had no evidence of endometriosis and 15 women who had laparoscopic evidence and histologically confirmed endometriosis were included in the study.

Endometrial cell culture

Endometrial and endometrioma biopsies were collected under sterile conditions and transported to the laboratory on ice in MEM{alpha} with 10% FBS. Normal endometrial stromal (NE) cells and eutopic endometriotic stromal (EE) cells from women with endometriosis and ectopic endometriotic stromal (EI) cells were prepared from biopsies and cultured under established conditions (25).

Briefly, the endometrial tissue was dissected free from underlying parenchyma, minced into small pieces, digested with collagenase (2 mg/ml) for 1 h at 37 C, and separated using serial filtration. Debris was removed by 100-µm aperture sieves, and epithelial glands were retained on 40-µm aperture sieves. Endometrial stromal cells in the filtrate were subcultured twice to eliminate contamination by macrophages and other leukocytes.

Extensive characterization of the cells confirmed that they retained phenotypic (e.g. IGF binding protein-1, vimentin) and functional (e.g. estrogen, progesterone, and epidermal growth factor receptors) markers of their endometrial and endometriotic origin in vivo (25). High purity (95%) of the stromal cell population was confirmed by negative staining with the following antibodies: CD3 (T cells), CD11b (granulocytes), CD45 (monocytes and other leukocytes), and cytokeratin (epithelial cells).

RANTES ELISA

Cell culture supernatants were collected and ELISA performed using a commercial human RANTES ELISA kit (Quantikine, R&D Systems). The samples and known RANTES standard protein preparations were incubated in 96-well plates precoated with monoclonal RANTES antibodies. All RANTES analyses were performed contemporaneously and in duplicate according to the manufacturer’s instructions.

Western blotting and EMSAs

Two independent methods were used to verify the functional activity of the NF-{kappa}B protein. Cell culture 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 centrifuged at 12,000 x g for 15 min at 4 C. Supernatants of extracts were denatured in Laemmli sample buffer, and 50 µg total protein were electrophoresed on 8% polyacrylamide SDS gels at 30 mA for 2 h. Proteins were transferred to 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 50 mM Tris, 150 mM NaCl, 0.1% Tween 20 containing 5% instant nonfat dry milk and then probed with 5 µg/ml of the anti-NF-{kappa}B p65 antibody at 4 C overnight. The antibody selectively binds the activated form of NF-{kappa}B. The polyvinylidene difluoride membranes were washed three times in the Tris-saline solution before the addition of a 1:5000 dilution of horseradish peroxidase-conjugated antimouse IgG, and specific bands were visualized by the enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL) method on X-OMAT film (Eastman Kodak Co., Rochester, NY). Differences in Western blots were estimated by transmission scanning using National Institutes of Health Image 1.60 software. All experiments were repeated twice to assure reproducibility.

EMSA was performed using standard reagents (Promega Corp., Madison, WI). NF-{kappa}B consensus oligonucleotides (5'-AGTTGAGGGGACTTTCCCAGGC-3'; 5'-GCCTGGGAAAGTCCCCTCAACT-3') were radiolabeled with [{alpha}-32P]ATP (3000 Ci/mmol, 10 mCi/ml) by using T4 polynucleotide kinase. The labeled oligonucleotides were separated from unincorporated nucleotides by chromatography through a G-25 spin column (Amersham Pharmacia Biotech, Piscataway, NJ). Retardation of proteins bound to oligonucleotides was determined by electrophoresis on a 6% polyacrylamide gel under nondenaturing conditions.

Reporter gene and expression vectors

The NF-{kappa}B response element reporter vector was a gift from Dr. Dale Leitman (Department of Obstetrics and Gynecology, University of California, San Francisco). The vector was constructed from the HIV-1 long terminal repeat and contains two binding sites for NF-{kappa}B, which are cloned in phase upstream of firefly luciferase. A 477-bp human RANTES promoter-luciferase fusion construct (–456/+20bp, relative to the transcription start site) was subcloned into the pGL2 vector (Promega). We previously determined that this construct contained the same transactivating function in endometrial stromal cells as the full-length (1 kb) RANTES promoter (8).

Transfection and cytokine treatment of endometrial stromal cells

Transient transfections were performed in human endometrial and endometriotic stromal cells grown in MEM{alpha} with 10% FBS and antibiotics in 12-well plates at about 50% confluence. pGL2–477-RANTES promoter, the NF-{kappa}B response element reporter construct, or pGL2-basic was added to each well using Effectene reagents (QIAGEN, Chatsworth, CA). In each case 0.45 µg plasmid DNA per well was added. The firefly luciferase reporter construct 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{alpha} with 10% FBS and antibiotics in the absence or presence of TNF{alpha} or IL-ß before luciferase activity was quantified. The concentrations of TNF{alpha} (2.9 nM) and IL-1ß (0.6 nM) used in their assays previously were shown to stimulate RANTES expression between 50 and 85% of maximal (8) (Hornung, D., I. Ryan, and R. N. Taylor, unpublished data). The results are presented as the percentage of luciferase activity between treated and untreated cells after correcting for transfection efficiency. Each reporter vector was assayed in three independent cultures. Empty pGL2-basic vector was analyzed as a control and revealed low basal activity as reported previously (8).

Data presentation and statistical analysis

RANTES ELISA data are presented as scatter grams of individual cell culture supernatants. Western blotting for activated NF-{kappa}B proteins and EMSA experiments were performed using extracts from three to four different cell preparations. Representative results are presented. RANTES promoter luciferase assay data are expressed as the mean ± SD of representative replicate experiments. Three independent experiments were performed with each promoter construct. Because the data did not show a Gaussian distribution and were expressed as a percentage of the controls, the results were compared with nonparametric Mann-Whitney U or Kruskal-Wallis tests using StatView software (SAS Institute, Inc., Cary, NC). Two-tailed tests with P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Secretion of RANTES protein in normal endometrial and ectopic endometriotic stromal cells

As we have reported previously, endometrial stromal cells grown in standard media do not secrete detectable RANTES protein. However, stromal cells isolated from women with ovarian EI showed higher levels of RANTES secretion after stimulation with TNF{alpha} (2.9 nM), compared with cytokine-stimulated NE stromal cells (Fig. 1Go) (P < 0.01).



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  		FIG. 1. Endometrial stromal cells isolated from women with ovarian endometriosis (EI cells; n = 9) showed higher levels of RANTES secretion after stimulation with TNF{alpha}, compared with NE stromal cells (n = 6) (P < 0.01).

 
Basal NF-{kappa}B activation and effect of sulindac on NF-{kappa}B translocation

Extracts from untreated NE and EI cells were analyzed by Western blotting to detect the activated form of NF-{kappa}B. Basal activation of NF-{kappa}B in EI cells was decreased after 1 h treatment with sulindac. TNF{alpha}, used as a positive control, increased NF-{kappa}B activation (Fig. 2Go).



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  		FIG. 2. Extracts from untreated EI cells were analyzed by Western blotting to detect the activated form of NF-{kappa}B. Basal activation of NF-{kappa}B in EI cells was noted (lane 1). Treatment with sulindac (300 µM) decreased the basal activation of NF-{kappa}B (lane 2), whereas TNF{alpha} (0.6 nM) increased its activation (lane 3).

 
To quantify NF-{kappa}B binding activity, nuclear extracts of NE, EE, and EI stromal cells were analyzed using EMSA. Specific protein-DNA complexes were induced by TNF{alpha} or IL-1ß after 1 h (Fig. 3Go) or 48 h (data not shown) under the same conditions. To verify the specificity of the binding reaction, we demonstrated that an excess of unlabeled oligonucleotide inhibited binding of the radiolabeled probe. Results for NE stromal cells are shown, but EE and EI stromal cell nuclear extracts gave similar responses. When NE stromal cells were preincubated with sulindac (800 µM) in the presence of TNF{alpha} (1.2 nM) or (IL)-1ß (0.6 nM), there was a dramatic decrease in cytokine-induced NF-{kappa}B binding activity after 1 h (Fig. 3Go) and 48 h (data not shown).



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  		FIG. 3. Activation of NF-{kappa}B was investigated by EMSA. Normal endometrial stromal cells were preincubated with sulindac (800 µM) in the presence or absence of TNF{alpha} (1.2 nM) or IL-1ß (0.6 nM). Nuclear extracts from untreated cells showed minimal NF-{kappa}B-binding activity, whereas specific complexes were induced by IL-1ß or TNF{alpha} after 1 h of treatment. To verify the specificity of the binding reaction, an excess of unlabeled oligonucleotide was added to the radiolabeled probe. Sulindac dramatically diminished TNF{alpha}- and IL-1ß-induced NF-{kappa}B binding activity after 1 h (lane 1: probe alone; lane 2: control; lane 3: control + unlabeled competitor probe; lane 4: IL-1ß; lane 5: sulindac; lane 6: TNF{alpha}; lane 7: sulindac/IL-1ß; lane 8: sulindac/TNF{alpha}. This example shows responses in normal endometrial stromal cells.

 
Effect of sulindac on NF-{kappa}B-mediated gene expression

Endometrial stromal cells were transiently transfected with a NF-{kappa}B response element construct. Treatment with TNF{alpha} (0.6 nM) induced a more than 2-fold increase in normalized luciferase activity, verifying that the regulation of the NF-{kappa}B promoter was intact in NE and EE stromal cells (Fig. 4Go, A and B). Pretreatment (4 h) with 300 µM sulindac decreased TNF{alpha}-induced NF-{kappa}B response element activity in NE and EE stromal cells by 81 and 94%, respectively (Fig. 4Go, A and B).



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  		FIG. 4. Endometrial stromal cells were transfected with an HIV-1 long terminal repeat luciferase construct containing two NF-{kappa}B-binding sites. Treatment with TNF{alpha} (0.6 nM) induced a more than 2-fold increase in normalized luciferase activity. Pretreatment (4 h) of NE and EE stromal cells with sulindac (Sul; 300 µM) decreased TNF{alpha}-induced NF-{kappa}B response element activity in NE (A) and EE (B) stromal cells by 81 and 94%, respectively. Asterisks (*) indicate a significant inhibition by Sul of the corresponding control or treatment (P < 0.05, Mann-Whitney test).

 
Effect of sulindac on TNF{alpha} stimulated RANTES gene transcription

To characterize the downstream effects of this robust NF-{kappa}B response, endometrial stromal cells were transiently transfected with the human RANTES gene promoter construct. Treatment with TNF{alpha} (0.6 nM) induced a more than 2-fold increase in normalized RANTES promoter luciferase activity in NE and EE stromal cells (Fig. 5Go, A and B). Pretreatment (4 h) with 300 µM sulindac decreased TNF{alpha}-induced RANTES promoter activation in NE stromal cells and EE stromal cells by 86 and 93%, respectively (Fig. 5Go, A and B).



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  		FIG. 5. Endometrial stromal cells were transfected with a –477 bp RANTES promoter construct. Treatment with TNF{alpha} (0.6 nM) induced a more than 2-fold increase in normalized luciferase activity. Pretreatment (4 h) of endometrial stromal cells sulindac (Sul; 300 µM) decreased RANTES promoter activation in normal endometrial cells in NE cells (A) and EE (B) stromal cells by 86 and 93%, respectively. Asterisks (*) indicate a significant inhibition by Sul of the corresponding control or treatment (P < 0.05, Mann-Whitney test).

 
Effects of sulindac on RANTES protein secretion

Endometrial stromal cells also secreted RANTES protein after treatment with TNF{alpha} (Fig. 6Go). The addition of sulindac to the endometrial stromal cells resulted in a significant suppression of TNF{alpha} and IL-1ß-induced RANTES protein secretion. In this example, TNF{alpha}- and IL-1ß-stimulated RANTES secretion by EI stromal cells was inhibited 61 and 79%, respectively, by sulindac (Fig. 6Go).



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  		FIG. 6. Treatment of EI stromal cells with IL-1ß (0.6 nM) or TNF{alpha} (1.2 nM) induced expression of RANTES protein. Pretreatment with sulindac (Sul; 300 µM) resulted in a significant decrease in RANTES secretion level by 61 and 79% by the two cytokines, respectively. Asterisks (*) indicate a significant inhibition by Sul of the corresponding control or treatment (P < 0.05, Mann-Whitney test).

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
We and others have postulated that endometriosis-associated symptoms are due to an aberrant inflammatory response in eutopic endometrium and within ectopic endometriotic tissues of women with this disease. Understanding the molecular mediators and pathways that underlie this response should provide clues to effective antiinflammatory therapies. Newer NSAIDs and COX-2 inhibitors, particularly sulindac and celecoxib, were shown to attenuate the implantation of eutopic endometrium and growth of implants in rodent models (26, 27). By contrast, the COX-2 inhibitor nimesulide failed to prevent the growth of human endometrial xenografts in athymic mice (28). To date, risks of thrombosis and myocardial infarction associated with highly selective COX-2 inhibitors have not been reported in patients using sulindac for joint pain (29). In the current study, we examined the antiinflammatory effects of sulindac on activation of the NF-{kappa}B pathway and RANTES gene and protein expression in normal endometrial and endometriotic stromal cells.

Inflammatory response is augmented in ectopic endometriotic stromal cells as shown by a significantly increased inducible RANTES secretion. Our finding corresponds to data showing that endometrial tissues and peritoneal macrophages derived from women with endometriosis exhibit an increased capacity to express other NF-{kappa}B target genes including IL-1ß, IL-6, IL-8, TNF{alpha}, and vascular endothelial growth factor (9, 30, 31, 32, 33, 34, 35). Several pathways activated by these cytokines, chemokines, and angiogenic factors might explain the increased inflammatory response in endometriosis. NF-{kappa}B, a key transcription factor, is an excellent potential candidate to target the inflammatory response in endometriotic cells (13, 14, 15, 18, 36). The physiological regulation of endometrial NF-{kappa}B during the normal menstrual cycle has not been extensively studied but appears to be expressed cyclically with peak levels of activation after ovulation (37) and during the perimenstrual phase (16). Progesterone, a physiological inhibitor of NF-{kappa}B in the uterus, suppresses NF-{kappa}B activity indirectly by stimulating synthesis of inhibitory-{kappa}B, the molecule that restrains NF-{kappa}B in the cytoplasm, and/or directly by inducing binding of progesterone receptor (PR) and NF-{kappa}B subunits (16, 18). Zhao et al. (20) and Sakamoto et al. (13) showed that the two major drugs used in the treatment of endometriosis, medroxyprogesterone acetate and GnRH agonist, both inhibit the NF-{kappa}B pathway in endometrial stromal cells. Such inhibition appears to suppress the expression of critical downstream effectors, like RANTES and IL-8. Furthermore, the NF-{kappa}B pathway was described to play a major role in estrogen-enhanced angiogenesis in endometrial adenocarcinoma cells (38).

Selective inhibition of NF-{kappa}B activation is an increasingly studied and sophisticated therapeutic approach for the treatment of several inflammatory disorders including rheumatoid arthritis, bronchial asthma, inflammatory bowel disease, and renal disease as well as some cancers (19, 39). Recently angiotensin-converting enzyme inhibitors were to shown to suppress induced release of cytokines (IL-8 and MCP-1) due to down-regulation of NF-{kappa}B activity (40). Sulindac is a potent NF-{kappa}B and nonselective COX inhibitor (21, 23) and is Food and Drug Administration approved for the treatment of rheumatoid arthritis, ankylosing spondylitis, and acute gout (41, 42, 43). Our experiments indicate that sulindac inhibits activation and DNA binding of NF-{kappa}B in endometrial stromal cells, supporting its antiinflammatory efficacy in these cells.

The presence of NF-{kappa}B response elements in the promoter region is a main feature of several inflammatory genes involved in the pathogenesis of endometriosis (19, 36). The RANTES promoter contains two NF-{kappa}B response elements proximal to the transcription start site (8). Site-directed mutagenesis of one NF-{kappa}B response element at –30 bp abolished the IL-1ß induction of RANTES gene expression in endometrial cells (8). In the current study, sulindac decreased RANTES gene expression and protein secretion in normal and endometriotic stromal cells. In this context, the suppressive effect of sulindac on RANTES expression is partly explained by the fact that sulindac prevents the shift of the NF-{kappa}B p65 subunit from the cytoplasm into the nucleus, resulting in decreased NF-{kappa}B mediated transcription. It was deduced previously that sulindac inhibits NF-{kappa}B by blocking inhibitory {kappa}-kinase and consequently preventing phosphorylation of inhibitory-{kappa}B{alpha} (21, 23).

Physiological suppression of NF-{kappa}B activity and associated cytokine expression by a functional PR is essential to the physiological regulation of inflammatory processes in the endometrium during the menstrual cycle (15, 16, 18, 44). Disturbance of antiinflammatory mechanisms leading to increased expression of proinflammatory cytokines in eutopic and ectopic endometrium of women with endometriosis is likely to involve both genetic and environmental factors. A preponderance of PR gene polymorphisms (+331G/A and PROGINS) in women with endometriosis lead to dysfunctional PR proteins (45, 46, 47). Polymorphisms in the PR gene also have been associated with a distorted PR-A to PR-B ratio (48), which could explain diminished PR mediated suppression of NF-{kappa}B-associated genes and supports the theory of progesterone resistance (49). A variety of environmental stimuli including nutritional factors such as coffee consumption (50) and inadequate intake of unsaturated fatty acids (51) and vegetables and fruits (52) also may promote inflammation by up-regulating genes such as RANTES, TNF{alpha}, IL-1ß, and IL-6. Moreover, dioxin, an environmental toxicant associated with the pathogenesis of endometriosis (53), was shown to induce RANTES expression in endometrial stromal cells (54). Dioxin also was shown to influence the PR-A to PR-B ratio in cocultures of endometrial stromal and epithelial cells (49) leading to a functional progesterone withdrawal. Whereas environmental factors such as dioxin and other arylhydrocarbons may play a pathogenic role in subsets of women with endometriosis, the interactions with NF-{kappa}B-regulated genes in women with endometriosis have yet to be defined.

Because NF-{kappa}B mediates inflammatory responses and cellular growth control (21), this pathway is a potential target in the pathogenesis of endometriosis. We have demonstrated that sulindac suppressed NF-{kappa}B activation, NF-{kappa}B-mediated gene transcription, RANTES gene expression, and protein secretion in endometrial stromal cells. Our results suggest that sulindac and potentially other NSAIDs and COX inhibitors, particularly those with strong anti-NF-{kappa}B activity, may serve as useful agents to reduce immune cell recruitment and activation in clinical endometriosis.


   Footnotes
 
This work was supported by Fonds zur Foerderung ver Wissenschaftlichen Forschung (FWF) Grant J2288-B13, National Institutes of Health Grant U54-HD37321, and a grant from Oracle, Giving (2004).

First Published Online September 13, 2005

Abbreviations: COX, Cyclooxygenase; EE, eutopic endometriotic stromal; EI, ectopic endometriotic stromal; FBS, fetal bovine serum; MCP, monocyte chemoattractant protein; NE, normal endometrial stromal; NF-{kappa}B, nuclear factor-{kappa}B; NSAID, nonsteroidal antiinflammatory drug; PR, progesterone receptor; RANTES, regulated on activation, normal T cell expressed and secreted.

Received May 3, 2005.

Accepted September 7, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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