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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1612
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 286-295
Copyright © 2005 by The Endocrine Society

Distinct Regulation of Cyclooxygenase-2 by Interleukin-1ß in Normal and Endometriotic Stromal Cells

Meng-Hsing Wu1, Chu-An Wang1, Chen-Chung Lin, Lei-Chin Chen, Wen-Chang Chang and Shaw-Jenq Tsai

The Institute of Clinical Medicine (M.-H.W.), The Institute of Molecular Medicine (C.-A.W., S.-J.T.), and Departments of Obstetrics and Gynecology (M.-H.W.), Physiology (C.-C.L., S.-J.T.), and Pharmacology (L.-C.C., W.-C.C.), College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan, Republic of China

Address all correspondence and requests for reprints to: Shaw-Jenq Tsai, Ph.D., Department of Physiology, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 70101, Taiwan, Republic of China. E-mail: seantsai{at}mail.ncku.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aberrant production of cyclooxygenase-2 (COX-2) plays pivotal roles in many pathological processes including tumorigenesis and endometriosis, although the underlying mechanism remains obscure. Herein we report evidence to demonstrate that COX-2 is distinctly regulated by IL-1ß in normal and endometriotic stroma. Ectopic endometriotic stromal cell is at least 100 times more sensitive to IL-1ß treatment, compared with its eutopic counterpart. Induction of COX-2 expression in normal endometrial stroma by IL-1ß is primary due to enhancement of COX-2 mRNA stability. In contrast, IL-1ß not only increases COX-2 mRNA stability but also up-regulates COX-2 promoter activity in ectopic endometriotic stroma. Induction of COX-2 promoter activity by IL-1ß is mediated via MAPK-dependent phosphorylation of cAMP-responding element binding protein. Promoter activity and EMSAs demonstrate that a cAMP response element site located at –571/–564 of COX-2 promoter is critical for IL-1ß-induced COX-2 gene expression. Our results indicate that elevation of COX-2 expression in endometriotic tissues may result from increased sensitivity to proinflammatory cytokines such as IL-1ß, which is consistently present in the peritoneal fluid of endometriosis patients. Distinct regulation of COX-2 gene by IL-1ß may play a critical role in pathophysiological processes such as cancer formation and endometriosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOMETRIOSIS IS A complex gynecological disorder with diverse sympotomatology, including dysmenorrhea, dyspareunia, pelvic pain, and infertility. Although multiple genetic and environmental factors seem to be important in the etiology of endometriosis (1), the critical factors that affect the implantation and propagation of endometriotic lesions are still largely unclear. Intrinsic molecular aberrations in pelvic endometriotic implants, compared with eutopic endometrium, have been proposed to be one critical factor leading to the development of endometriosis. Because endometriosis is highly estrogen dependent, aberrant expression of steroidogenic acute regulatory protein and aromatase in endometriotic implants resulting in overproduction of 17ß-estradiol have been postulated to play pivotal roles in the development of endometriosis (2, 3, 4). Recent studies (2, 5, 6) demonstrate that aberrant expression of steroidogenic acute regulatory protein and aromatase in endometriotic stromal cells is stimulated by the same molecule, prostaglandin (PG) E2. Increased concentrations of PGE2 in the peritoneal fluid of patients with endometriosis and the mechanisms of PGE2 actions in regulation of steroidogenesis have been reported (5, 6, 7, 8). However, the cellular origin of PGE2 in endometriosis was not clear by far.

It is generally believed that PGE2 must be produced and functions locally because PGs are unstable eicosanoids with very short half-life (9). Indeed, peritoneal macrophages obtained from patients with endometriosis are known to have greater PG synthetic capability, compared with those from endometriosis-free women (8). An alternative but not necessarily mutually exclusive hypothesis is PGE2 may be synthesized by pelvic endometriotic implant because eutopic endometrium is capable of producing PGs during normal menstrual cycle. In agreement with this hypothesis, elevated expression of cyclooxygenase (COX)-2 in ectopic endometriotic lesion was detected (10, 11). However, the mechanism responsible for elevation of COX-2 in ectopic endometriotic implants remains uncharacterized.

COX, also called PG G/H synthase, is the first rate-limiting enzyme in the biosynthesis of PGs (including PGE2), controlling the conversion of arachidonic acid to PGH2 (the common precursor for PGs). Currently, there are three known isoforms of COX enzyme, COX-1, COX-2, and COX-3 (a splice variant of COX-1) (12, 13). COX-1 is expressed in many tissues under basal conditions and is thought to be responsible for producing PGs for primary housekeeping functions such as platelet aggregation, vasodilatation in the kidney, and cytoprotection of gastric mucosa (12, 14). In contrast, COX-2 is usually absent and is induced in many different cell types after diverse stimulation, including mitogens, growth factors, cytokines, proinflammatory agents, and tumor promoters (12, 15, 16). Thus, PGs produced by COX-2 up-regulation usually leads to pathological alteration in various tissues, and determination of COX-2 overexpression may reveal information related to disease status.

There is local, sterile inflammation in the peritoneal cavity of the patients with endometriosis, and increased proinflammatory agents in peritoneal fluid such as IL-1ß has been reported (17, 18). These cytokines are polypeptides or glycoproteins acting as autocrine/paracrine signals to regulate immune response and inflammation. They contribute to the formation of endometriosis through influencing the establishment and proliferation of ectopic endometrial implants and further cytokine secretion by macrophages (17). These proinflammatory cytokines represent likely candidates for stimulation of COX-2 expression in ectopic endometriotic implant and peritoneal macrophage. The objective of the current study was to investigate mechanisms responsible for elevated expression of COX-2 in ectopic endometriotic tissue. Our results demonstrate that COX-2 gene is at least 100 times more sensitive to IL-1ß treatment in ectopic endometriotic stroma as compared with its eutopic counterpart. Increased sensitivity of COX-2 gene to IL-1ß may be one reason of sustained elevation of COX-2 protein in ectopic endometriotic lesion, given that significant concentration of IL-1ß is consistently present in the peritoneal fluid of patients with endometriosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and reagents

All chemicals used in this study, unless otherwise specified, were purchased from Sigma Chemical Co. (St. Louis, MO). Taq DNA polymerase, fetal bovine serum, DMEM/F-12, antibiotics, and 1 kb DNA ladders were from Gibco/BRL (Gaithersburg, MD). Magnetight oligo(dT) particles were from Novagen (Madison, WI). Polyvinylidene difluoride membrane was from Millipore Co. (Bedford, MA). Western blot chemiluminescence reagents were from NEN Life Science Products Life (Boston, MA). SB202190, BAY 11–7802, U0126, and SP600125 were from Calbiochem (San Diego, CA). Mouse anti-COX-2 monoclonal antibody, rabbit-anti-COX-1 polyclonal antiserum, and a PGE2 enzyme immunoassay (EIA) kit were purchased from Cayman Biochemical Co. (Ann Arbor, MI). Anti-ERK1/2, antiphospho ERK1/2 (Thr202/Tyr204), anti-p38 MAPK, antiphospho p38 MAPK (Thr180/Tyr182), anti-c-Jun N-terminal kinase (JNK), and antiphospho JNK (Thr183/Tyr185) were from Cell Signaling Technology (Beverly, MA). The antibodies against cAMP response element-binding protein (CREB) and phosphorylated CREB (Ser133) were from Upstate Inc. (Waltham, MA). IL-1R1 and nuclear factor-{kappa}B (NF-{kappa}B) antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), whereas anti-IL-1R2 antibody was from R&D Research (San Diego, CA).

Collection of tissues

The subjects consisted of 40 women who were treated at the Department of Obstetrics and Gynecology at National Cheng Kung University Hospital. The use of these tissues was approved by the Clinical Research Ethics Committee at the National Cheng Kung University Medical Center, and informed consent was obtained from each patient. During the time of laparoscopy or laparotomy, the ovarian endometrioma (from severe endometriosis, n = 12), pelvic endometriotic implants (from early endometriosis, n = 10), and normal endometria (from disease-free patients, n = 13) were collected. In addition, five sets of samples consisting of ovarian endometrioma and the eutopic endometria were also collected from patients with severe endometriosis according to the revised American Society of Reproductive Medicine classification (1997). Tissues were immediately brought to the laboratory on ice in sterile tubes containing PBS. Half of the tissues were frozen in liquid nitrogen and stored at –80 C until used. The other half of tissues were minced and subjected to further isolation of stromal cells.

Isolation and culture of stromal cells

The procedure used to isolate stromal cells from uterine endometrium and ectopic endometriotic implant was described previously (2, 19, 20). Purity of the cell was immunostained with vimentin (stromal cell specific) and cytokeratin (epithelial cell specific). Originality of endometriotic stromal cells was confirmed by measurement of prolactin production after induced decidualization in vitro as previously described (20). Stromal cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum and antibiotics at 37 C (2 x 104 cells/well for RNA isolation; 5 x 105 cells/dish for protein isolation) until 70% confluence was reached. After serum starvation for 12 h, the cells were stimulated with IL-1ß (0.01–10 ng/ml) for 12 h. For mRNA isolation, cells were directly lysed in the well using lysis buffer. For protein isolation, cells were lysed and collected in the dish using radioimmunoprecipitation assay buffer (described below). In a separate experiment, cells were treated with several inhibitors including PD098059 or U0126 [MAPK kinase (MEK) inhibitors], BAY11–7081 [I{kappa}B kinase (IKK) inhibitor], SP600125 (JNK inhibitor), and SB 202190 (p38 MAPK inhibitor) with or without 1 ng/ml IL-1ß for different lengths of time as described in the figure legends. Cells were lysed for mRNA or protein analysis, and culture media were collected for PGE2 determination. For mRNA stability experiment, cells were pretreated with TNF{alpha} (1 pg/ml) for 2 h to up-regulate the expression of COX-2 transcript. TNF{alpha} was then removed by replacing with fresh serum-free medium containing actinomycin D (1 µM). After 30 min, IL-1ß (1 ng/ml) was added to the culture medium, and cells were harvested at various time points.

RNA isolation, RT-PCR, and real-time RT-PCR

Total RNA was isolated from normal endometria and endometriotic biopsies using Rneasy mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Poly (A+) RNA was isolated from cultured stromal cells using Magnetight oligo(dT) beads as previously described (2, 21). The mRNA concentrations were determined by the UV absorption at 260 nm. RNA samples (500 ng total RNA or 50 ng mRNA) were reversed transcribed at 42 C for 60 min and 2 µl of reverse transcription products were then subjected to PCR amplification using an ABI 7900 thermal cycler (Applied Biosystems, Foster City, CA). Simple RT-PCR was performed to detect receptors of IL-1ß. The sequences of primers used were as follows: IL-1R1, 5'-TACTTGGGCAAGCAATATCC-3' and 5'-GCGTCATAGGTCTTTCCATC-3'; IL-1R2, 5'-AAGGCCAGCAATACAACATC-3' and 5'-CGTCTGTGCATCCATATTCC-3'; and IL-1Ra, 5'-GGGTGCTACTTTATGGGCAG-3' and 5'-TCGTCAGGCATATTGGTGAG-3'. The primer sequences for glyceraldehyde-3-phosphate dehydrogenase were reported previously (23). The cycling conditions were 95 C for 10 min, followed by 30 cycles of 95 C for 30 sec, 57 C for 30 sec, and 72 C for 30 sec.

To determine the absolute amount of COX-2 transcripts in the mRNA stability experiment, real-time RT-PCR was conducted. In this reaction, SYBR Green I was added to the PCR master mix and served as a fluorescence source for laser detection. The in vitro-transcribed COX-2 RNA as previously described (23) was serially diluted (0.01–1000 amol) and used to generate a standard curve. Nucleotide sequences of the primers were: COX-2, 5'-AGGGCCAGCTTTCACCAAC-3' and 5'-AAGGCGCAGTTTACGCTGTC-3' and 18S rRNA, 5'-GTGTGCCTACCTACG-3' and 5'-TGACCCGCACTTACTG-3'. The cycling conditions were 95 C for 10 min, followed by 40 cycles of 95 C for 30 sec, 59 C for 30 sec, and 72 C for 30 sec. The reaction data were expressed as attomoles per microgram RNA after converting the number of cycle thresholds, which is the PCR cycle number at which the fluorescent signal in each reaction reaches a preset threshold above background, to absolute value according to the standard curve. A dissociation curve was created using the built-in melting curve program to confirm the presence of a single PCR product.

Western blotting

Tissues and treated stromal cells were homogenized under radioimmunoprecipitation assay buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate in PBS buffer) and centrifuged at 600 x g for 30 min at 4 C to remove debris. Equal amount of protein was loaded into each lane, separated on a 10% SDS-PAGE, and transferred onto a polyvinyl difluoride membrane. Detail procedure for Western blotting was described previously (23).

PGE2 assay

To determine the concentrations of PGE2, cultured media were collected and subjected to PGE2 measurement. PGE2 was determined using an EIA kit according to the procedure provided by the manufacturer. The sensitivity (80% bound) was 15 pg/ml with intra- and interassay coefficients of variant of 4.6 and 9.2%, respectively.

Plasmids, transfection, and promoter activity assays

The upstream region (–918/+49) of the human COX-2 promoter was cloned to the pXP2 vector containing the luciferase reporter system. Serial deletion constructs were generated from the –918/+49 pXP2 plasmid using a PCR amplification approach. A commercial plasmid containing cytomegalovirus-driven Renilla reporter system was purchased from Promega Corp. (Madison, WI). Cells were plated on 24-well plates for the luciferase/Renilla assays. Plasmids were transfected using lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA). Transfections were followed by rising and incubation in serum-free DMEM/F12 medium for 12 h. After medium was changed, cells were then treated with IL-1ß (0.1, 1, or 10 ng/ml) for another 12 h in the presence or absence of different kinase inhibitors. Luciferase assays were performed using the dual luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Briefly, 100 µl luciferase substrates were added to 20 µl lysate, and luciferase activity was measured using a 20/20 luminometer (Turner Designs, Sunnyvale, CA). Each luciferase assay experiment was performed in triplicate and repeated for the number of times indicated in the figure legends using different batches of cells.

EMSA

Double-stranded oligonucleotides corresponding to cAMP response element (CRE) binding sites (–580 to –556) in the human COX-2 promoter were synthesized and annealed in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 25 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol. The positive strand of oligonucleotide probes was labeled with carboxyfluorescein fluorescence dye for the detection of fluorescent intensity. Unlabeled consensus CRE (50-fold excess, Promega) was used as competitors in some experiments. A total of 15–45 µg nuclear extract from ectopic endometriotic stromal cells was incubated in the presence or absence of competitor for 20 min at 10 C in binding buffer. The DNA/protein complexes were resolved on a 5% nondenaturing acrylamide gel in a buffer of 0.25 M Tris, 1.9 M glycine, and 10 mM EDTA final, which was exposed to fluorescent phosphor imager FLA3000 (Fuji, Tokyo, Japan).

Statistical analysis

All data are expressed as mean ± SEM. Data were analyzed by one-way ANOVA followed by Tukey’s test if significant differences were found. Significant differences were accepted when two-tailed analysis yielded P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
COX-2 is overexpressed in ectopic endometriotic implants

To investigate whether elevated concentration of PGE2 in peritoneal fluid of women with endometriosis is contributed by ectopic endometriotic implants, tissues obtained from normal endometrium and ectopic endometriotic lesion were used to quantify concentrations of COX-2, the key enzyme to control PGE2 production. Ectopic endometriotic implants obtained from patients with pelvic implant and ovarian endometrioma expressed much greater COX-2 mRNA and protein than eutopic endometrium of endometriosis-free women (Fig. 1Go, A and B). On the other hand, expression of COX-1 in both eutopic and ectopic endometrial tissues was minimal, and no substantial change was observed (Fig. 1BGo). Analysis of freshly isolated endometrial stromal cells obtained from paired eutopic and ectopic endometrial tissues of women with endometriosis (n = 5) demonstrated that COX-2 protein was markedly expressed in ectopic endometriotic stromal cells (Fig. 1CGo). Consequently, PGE2 production was significantly increased in stromal cells derived from ectopic endometriotic implant, compared with that from normal endometrium (Fig. 1DGo).



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  		FIG. 1. Expression of COX-2 in ectopic endometriotic stromal cells is more pronounced than that in eutopic endometrial stromal cells. A, COX-2 mRNA levels in pelvic endometriotic implants (PE, n = 10), ovarian endometriomas (OvE, n = 12), and normal endometrium (N, n = 13) were determined by real-time RT-PCR. Data are means ± SEM, and asterisks denote significant difference from normal group (P < 0.05). B, A representative Western blot showing that the expression of COX-2 but not COX-1 protein is markedly increased in endometriotic tissues from patients with endometriosis. Eight samples from each group were determined, and the results were similar. C, A representative Western blot demonstrates that marked elevation of COX-2 protein expression in freshly isolated ectopic (Ec) endometrial stromal cells, compared with that in eutopic (Eu) endometrial stromal cells. S1, S2, and S3 denote different patients. Five matched pairs of eutopic and ectopic endometrial stromal cells were determined with identical results. D, Production of PGE2 by stromal cells isolated from ectopic endometriotic tissue (E) and normal endometrium (N) was determined by EIA. Data are means ± SEM from seven independent experiments with different batches of cells. Asterisk denotes significant difference between endometriosis and normal group (P < 0.05).

 
Effect of IL-1ß on COX-2 expression

To determine whether aberrant COX-2 expression in endometriotic tissues was due to stimulation by IL-1ß, stromal cells isolated from endometriotic implants were subjected to various IL-1ß treatment regimens. Dose-effect studies demonstrated that both COX-2 mRNA and protein were induced by IL-1ß in a dose-dependent manner (Fig. 2Go, A–C), which resulted in concomitant elevation of PGE2 concentrations in conditioned media (Fig. 2DGo). Administration of IL-1ß (1 ng/ml) caused a marked increase in COX-2 expression by ectopic endometrial stromal cells in a time-dependent manner, which peaked at 8–12 h from treatment (Fig. 2EGo). To confirm that IL-1ß-induced PGE2 production is controlled by up-regulation of COX-2 but not COX-1, a selective COX-2 inhibitor, NS398, was used to blunt COX-2 activity. As shown in Fig. 2FGo, pretreatment with NS398 completely blocked the IL-1ß-induced PGE2 production by ectopic endometrial stromal cells without affecting COX-2 protein expression (Fig. 2GGo).



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  		FIG. 2. IL-1ß induces COX-2 mRNA and protein expression in ectopic endometriotic stromal cells. A–C, The mRNA and protein of COX-2 are dose-dependently induced by IL-1ß in cultured endometriotic stromal cells. D, Production of PGE2 by endometriotic stromal cells is also dose-dependently stimulated by IL-1ß. Data are means ± SEM from six experiments. The asterisks denote significant difference from no IL-1ß treatment (0) group (P < 0.05). E, A representative Western blot demonstrates that COX-2 expression induced by IL-1ß (1 ng/ml) is time dependent. Four independent experiments were performed using different batches of cells, and the results were similar. F and G, Pretreatment with COX-2 inhibitor, NS398 (1 µM), completely inhibits PGE2 production induced by IL-1ß but does not affect the expression of COX-2 protein. F, Means ± SEM of six independent experiments. Asterisk denotes significant difference from control (con) (P < 0.05).

 
IL-1ß induces more COX-2 expression in ectopic endometriotic stromal cells

To test whether IL-1ß exerts different actions in endometria of normal and patients with endometriosis, stromal cells obtained from normal endometria or endometriotic implants were treated with IL-1ß, and COX-2 expression was determined. In agreement with previous results, the basal level of COX-2 in stromal cells derived from endometriosis was greater than that in stromal cells from normal endometrium (Fig. 3AGo). Notably, COX-2 protein was induced by treatment with IL-1ß in both eutopic and ectopic endometrial stromal cells, but the degree of induction was dramatically different (Fig. 3AGo). Expression of COX-2 in ectopic endometriotic stromal cell was at least 10-fold greater than that in endometrial stromal cell when IL-1ß was applied (Fig. 3AGo).



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  		FIG. 3. IL-1ß induces much greater COX-2 expression in ectopic endometriotic stromal cells. A, A representative Western blot shows the induction of COX-2 protein by IL-1ß in normal endometrial stromal cells and endometriotic stromal cells (upper panel). The lower panel shows means ± SEM from seven experiments. Asterisks denote significant difference from normal control (con) (P < 0.05), whereas the # sign indicates a difference between IL-1ß-treated normal and endometriotic stromal cells (P < 0.05). B, A representative Western blot of one pair of eutopic and ectopic endometrial stromal cells obtained from a patient with endometriosis treated with different concentrations of IL-1ß. Five pairs of eutopic and ectopic endometrial stromal cells were used to repeat this experiment with similar results. C, Mean values of PGE2 produced by stromal cells treated with or without IL-1ß from five sets of matched pair samples. Asterisks denote significant difference from normal control, whereas the # sign indicates difference between IL-1ß-treated normal and endometriotic stromal cells (P < 0.05).

 
Because the normal endometrial stromal cells and ectopic endometriotic stromal cells were obtained from different subjects, it is possible that differential responses to IL-1ß may be due to a difference in genetic background. To investigate this possibility, stromal cells from paired eutopic and ectopic tissues (n = 5) originally obtained from patients with severe endometriosis were used. Stromal cells derived from eutopic endometrium of patients with endometriosis expressed minute amounts of COX-2, which is not different from stromal cells obtained from the endometrium of women without endometriosis (Fig. 3Go, A and B). The stromal cells obtained from ectopic endometriotic tissues had higher basal levels of COX-2 protein and were more responsive to IL-1ß (Fig. 3Go, A and B). Treatment with 0.1 ng/ml IL-1ß induced much greater COX-2 expression in ectopic endometriotic stromal cells, compared with that in eutopic endometrial stromal cells. The induction was even more pronounced than that in eutopic endometrial cells treated with 10 ng/ml IL-1ß. In concordance with this result, production of PGE2 by ectopic endometriotic stromal cells treated with IL-1ß was 10–20 times greater, compared with its eutopic counterpart (Fig. 3CGo).

Increased sensitivity to IL-1ß in ectopic endometriotic stromal cells is not due to increased IL-1ß receptors and/or COX-2 mRNA stability

In seeking factors leading to distinct responsiveness to IL-1ß, we first decided to evaluate the concentrations of IL-1 receptors (IL-1R1) and its decoy receptor (IL-1R2) as well as the antagonized receptor (IL-1Ra) in stromal cells of eutopic and ectopic endometria. By using RT-PCR, concentrations of mRNA encoding for IL-1R1 were not different between normal and endometriotic stroma although different levels of expression were observed between individuals (Fig. 4AGo). In contrast, the mRNA encoding for IL-1R2 and IL-1Ra was undetectable under our condition (Fig. 4AGo). By increasing PCR cycle number to 35 cycles, we were able to detect faint bands corresponding to IL-1R2 amplified from both eutopic and ectopic samples, but the intensity was no different (data not shown). Western blot analysis demonstrated that expression of IL-1R1 and IL-1R2 between stromal cells of eutopic and ectopic origins was not different at the protein level (Fig. 4BGo). In addition, treatment with IL-1ß did not significantly affect the level of its receptors (Fig. 4BGo).



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  		FIG. 4. Expression of IL-1 receptor mRNA and protein in normal and ectopic endometrial stromal cells. A, Expression of mRNA encoding for IL-1R1 (R1), IL-1R2 (R2), and IL-1Ra (Ra) was determined by RT-PCR. The mRNA obtained from the macrophage was used as positive control (PC) for R2 and Ra, whereas glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for internal control. S1-S5 denote five individuals with severe endometriosis from which eutopic (Eu) and ectopic (Ec) endometrial stromal cells were purified. B, A representative Western blot of IL-1R1 and IL-1R2 protein in eutopic (Eu) and ectopic (Ec) endometrial stromal cells treated or not treated with IL-1ß (1 ng/ml) for 12 h. Five sets of paired eutopic and ectopic samples were used to repeat this experiment, and the results were similar.

 
Another possibility that may lead to distinct induction of COX-2 by IL-1ß is different signaling pathway being used in eutopic and ectopic endometrial stroma. To address this possibility, phosphorylation and/or nuclear translocation of major signaling molecules downstream of IL-1ß were examined. Treatment with IL-1ß led to rapid phosphorylation of ERK, p38 MAPK, and JNK, which was again no different between eutopic and ectopic endometrial stroma (Fig. 5AGo). Activation and nuclear translocation of NF-{kappa}B is another important signaling cascade that mediates IL-1ß action. As shown in Fig. 5BGo, IL-1ß induced rapid nuclear translocation of NF-{kappa}B. Again, there was no statistical difference in amounts of NF-{kappa}B translocated to nucleus between eutopic and ectopic endometrial stroma (Fig. 5BGo).



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  		FIG. 5. Expression of COX-2 induced by IL-1ß is mediated via multiple signaling cascades. A, Eutopic and ectopic endometrial stromal cells were treated with IL-1ß (1 ng/ml) for 15, 30, or 60 min, and phosphorylation of ERK, p38 MAPK, and JNK was determined. This experiment was repeated three times with similar result. B, Eutopic and ectopic endometrial stromal cells were treated with IL-1ß (1 ng/ml) for 15 or 30 min, and activation of NF-{kappa}B was determined by blotting the p65 subunit in nuclear fraction. This experiment was repeated three times, and the results were similar. C, Paired eutopic and ectopic endometrial stromal cells were pretreated with MEK (U0, 10 µM), p38 MAPK (SB, 10 µM), IKK (BAY, 5 µM), or JNK (SP, 10 µM) inhibitors 30 min before addition of IL-1ß (1 ng/ml), and COX-2 protein was detected at 12 h after IL-1ß treatment. This experiment was repeated three times using paired samples and four more times with unpaired normal endometrial stromal cells and endometriotic stromal cells (data not shown), and the results were similar. Con, Control.

 
To explore which signaling cascade indeed mediates IL-1ß-induced COX-2 expression, selective inhibitors of various protein kinases downstream of IL-1ß signaling were administered. Treatment with BAY 11–7081 (disrupts NF-{kappa}B pathway), U0126 (disrupts ERK pathway), and SB202190 (disrupts p38 MAPK pathway) decreased IL-1ß-induced COX-2 protein expression (Fig. 5CGo). In contrast, treatment with SP600125 (disrupts JNK pathway) failed to affect COX-2 protein expression, suggesting that although JNK was phosphorylated by IL-1ß treatment, its activation might not be involved in COX-2 expression. The inhibition percentage contributed by disrupting NF-{kappa}B and p38 MAPK signaling pathways was similar between eutopic and ectopic endometrial stromal cells (Fig. 5CGo). Interestingly, administration of U0126 caused more pronounced inhibitory effect on ectopic endometriotic stromal than on eutopic endometrial stroma (Fig. 5CGo), indicating potential distinct effects of ERK signaling on these two cell lineages.

We next decided to test whether differences in COX-2 expression were due to distinct effects of IL-1ß on increasing COX-2 mRNA stability. By using real-time quantitative RT-PCR, we found the initial amount of COX-2 transcripts at time 0 (time of IL-1ß addition) was 20–30 times greater than that in eutopic endometrial stoma (55.6 ± 7.5 vs. 2.3 ± 0.6 amol/µg RNA for ectopic and eutopic stroma, respectively). Despite the great difference in initial amounts of transcripts, the half-life of COX-2 mRNA (about 1.5 h) in ectopic endometriotic stroma was not different from that in eutopic endometrial stroma (Fig. 6AGo). Treatment with IL-1ß significantly enhanced COX-2 mRNA stability, which was evident in both eutopic and ectopic endometrial stromal cells (Fig. 6AGo). Nevertheless, no differential effect on enhancing COX-2 mRNA stability between eutopic and ectopic endometrial stroma was observed (Fig. 6AGo).



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  		FIG. 6. IL-1ß enhances COX-2 mRNA stability in both eutopic and ectopic endometrial stromal cells. A, Stromal cells obtained from normal and endometriotic tissues were pretreated with TNF{alpha} (1 pg/ml) for 2 h to induce expression of COX-2 transcripts. After washing off TNF{alpha}, cells were incubated in fresh medium containing 1 µM actinomycin D for 30 min and then treated with IL-1ß (1 ng/ml) for 1, 2, 4, or 8 h. Concentrations of mRNA were quantified using real-time RT-PCR with external standards, and values at time 0 (the point of IL-1ß addition) were normalized to 100%. Asterisk denotes significant difference from corresponding controls (Con) (without IL-1ß treatment) by repeat-measured-one way ANOVA. B, Cells were treated as mentioned above and selective inhibitors (U0126, 10 µM; SB, 10 µM; BAY, 5 µM, and SP, 10 µM) were added 30 min before IL-1ß treatment. At 4 h after IL-1ß treatment, cells were harvested and mRNA was isolated. COX-2 transcripts were quantified by real-time RT-PCR, and the values at time 0 were normalized to 100%. Data are means ± SEM from four independent experiments. Asterisk denotes significant difference from IL-1ß-treated group. con, Control.

 
Effects of different signal pathways on COX-2 mRNA stability were further evaluated by quantification of COX-2 transcripts at 4 h after IL-1ß treatment. Pretreatment with U0126, BAY 11–7081, and SB202190 significantly attenuated IL-1ß-induced COX-2 mRNA stability, whereas SP600125 had no substantial effect (Fig. 6BGo). However, the inhibitory effect of U0126, BAY 11–7081, and SB202190 was again similar between eutopic and ectopic endometrial stroma (Fig. 6BGo).

IL-1ß induces up-regulation of COX-2 promoter activity in ectopic endometriotic stromal cells

We next sought to determine whether a distinct effect of IL-1ß on COX-2 expression is regulated at the transcriptional level. A human COX-2 promoter (–918/+49) was cloned to luciferase reporter plasmid and transiently transfected into normal endometrial stromal cells or ectopic endometriotic stromal cells. Treatment with IL-1ß had no substantial effect on COX-2 promoter transfected into normal endometrial stromal cells (Fig. 7AGo). In contrast, IL-1ß dose-dependently up-regulated COX-2 promoter activity when it was transfected to stromal cells derived from ectopic endometriotic tissue (Fig. 7AGo).



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  		FIG. 7. IL-1ß induces COX-2 promoter activity in stromal cells of endometriotic tissues is mediated by ERK and p38 MAPK signaling cascades. A, Constructs of the COX-2 promoter (–918/+49) linked to the luciferase reporter were transiently transfected into eutopic and ectopic endometrial stromal and stimulated with or without IL-1ß for 12 h, and then luciferase activity was analyzed. The promoter activity (RLU) is calculated by dividing firefly signals to Renilla signals. Due to variations between individuals, value of the control group (without IL-1ß treatment) was normalized to 1. Asterisks denote significant difference from control group (0). B, Ectopic endometriotic stromal cells were transfected with COX-2 promoter construct (–918/+49), treated with IL-1ß (1 ng/ml) for 12 h in the presence or absence of different selective inhibitors, and then luciferase activity was analyzed. The asterisk denotes the significant difference from IL-1ß-treated group. *, P < 0.05; **, P < 0.001. C, Schematic drawing of annotated human COX-2 promoter with important putative transcription factor binding sites. The predicted CRE (WtCRE) and site-mutated CRE (mCRE) sequences were listed. Arrow indicates the 5' end of each construct. D, Serial deletion constructs of the COX-2 promoter linked to the luciferase reporter were transiently transfected into eutopic and ectopic endometrial stromal and stimulated with or without IL-1ß for 12 h, and then luciferase activity was analyzed. Data are means ± SEM from four experiments using different matched pairs of cells. The asterisk denotes significant difference between untreated and IL-1ß-treated groups of each corresponding construct. Con, Control; GAS, interferon {alpha}-activated sequence; Sp1, SV40 promoter-1; C/EBP, CCAAT/enhancer-binding protein.

 
Next, selective inhibitors were applied to transiently transfected stromal cells to determine which signaling pathway is involved in IL-1ß-induced COX-2 promoter activity. As shown in Fig. 7BGo, blockage of MEK activity completely blocked IL-1ß-induced COX-2 promoter activity, whereas disruption of p38 MAPK by SB202190 resulted in only partial inhibition. In contrast, SP600125 and BAY 11–7081 had no effect on inhibition of IL-1ß-induced COX-2 promoter activity (Fig. 7BGo). This result further confirmed the data that U0126 reduced more IL-1ß-induced COX-2 protein expression than other inhibitors in ectopic endometriotic stroma (Fig. 5CGo). To identify the region of the COX-2 promoter responsible for inductive effects of IL-1ß, bioinformatic analysis was performed to annotate the human COX-2 promoter up to –918 bp. Several putative response elements were annotated within this region (Fig. 7CGo), and a series of deletion constructs corresponding to remove each of these elements were generated. Transient transfections were performed using these constructs, and the corresponding promoter activities were determined. As expected, IL-1ß failed to induce promoter activity of any COX-2 reporter constructs transfected into normal endometrial stromal cells (Fig. 7DGo). In ectopic endometriotic stromal cells, deletion of the –918 to –663 bp had no substantial effect on IL-1ß-induced COX-2 promoter activity. Deletion of the –663 to –550 bp completely abolished IL-1ß-induced COX-2 promoter activity (Fig. 7DGo), whereas deletion to –80 bp further reduced basal COX-2 promoter activity (Fig. 7DGo). The result indicates that the CRE lying between –663 and –550 bp is necessary for IL-1ß-mediated COX-2 induction in ectopic endometriotic stromal cells.

CREB and IL-1ß-induced COX-2 promoter activity

To further confirm that the CRE located at –571/–564 is important for IL-1ß-induced COX-2 promoter activity, site-mutated CRE was used for promoter activity assay. As shown in Fig. 8AGo, mutagenesis of CRE completely abrogated IL-1ß-induced COX-2 promoter activity. Another piece of evidence showing that CRE is important for COX-2 promoter activity was brought by EMSA. Binding of CREB to fluoresce-labeled synthetic oligo-probe identical with putative CRE sequence and flanking region at –580/–556 of COX-2 promoter was evident by EMSA (Fig. 8BGo). The binding complex was demolished by adding excess unlabeled consensus CRE oligo (Promega) but not mutated oligo (Fig. 8BGo).



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  		FIG. 8. CREB is critical for IL-1ß-induced human COX-2 promoter activity in ectopic endometriotic stromal cell. A, Ectopic endometriotic stromal cells were transiently transfected with COX-2 promoter constructs (–918/+49) containing intact (wt CRE) or site-mutated CRE (mCRE) and treated with IL-1ß (1 ng/ml) for 12 h, and then luciferase activity was analyzed. Data are means ± SEM from four independent experiments. Asterisk denotes significant difference from IL-1ß-treated group (P < 0.05). B, Nuclear extracts (N.E.) isolated from IL-1ß-treated ectopic stromal cells were incubated with FAM-labeled oligonucleotide corresponding to putative CRE site and flanking region of COX-2 promoter region (–580/–556) and subjected to electrophoresis and detection. Triangle indicates different amounts of nuclear extract (15, 30, and 45 µg protein from left to right) added. Identical results were obtained from four independent experiments using different batches of cells. C, Ectopic endometriotic stromal cells were treated with IL-1ß (1 ng/ml) for 15 min in the presence or absence of U0126 (U0), SB202190 (SB), BAY 11–7081 (Bay), or SP600125 (SP), and the phosphorylation of CREB was determined. This experiment was repeated four times using different batches of cells with similar results. Con, Control.

 
By far, our evidence suggests CREB is involved in IL-1ß-induced COX-2 promoter activity. To further verify this indeed mediates elevation of COX-2 expression in ectopic endometriotic stroma, we then detected the phosphorylation of CREB after IL-1ß treatment. Increased phosphorylation of CREB was evident at 15 min after IL-1ß treatment (Fig. 8CGo). Administration of U0126 significantly inhibited IL-1ß-induced CREB phosphorylation (Fig. 8CGo). In contrast, cotreatment with BAY 11–7081 or SP600125 failed to inhibit IL-1ß-induced CREB phosphorylation (Fig. 8CGo). These data provide further evidence to support the notion that IL-1ß-induced COX-2 expression is mediated via ERK-dependent CREB phosphorylation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Elevated concentration of PGE2 in the peritoneal fluid of women with endometriosis is an important factor contributing to the development and symptomatic consequences of endometriosis. Aberrant expression of COX-2 in peritoneal macrophage and ectopic endometriotic implant has been reported, although the underlying mechanism remains obscure (8, 10). In this work, we identify that expression of COX-2 in eutopic endometrial and ectopic endometriotic stromal cells is distinctly regulated by IL-1ß. The induction of COX-2 by IL-1ß in ectopic endometriotic stromal cell is at least 100 times more sensitive, compared with its eutopic counterpart. Notably, increased sensitivity of IL-1ß-induced COX-2 expression is mediated via increasing COX-2 promoter activity governed mainly by MAPK-dependent signaling through binding to the CRE site at –571/–564 of the COX-2 promoter. To our knowledge, this is the first report that clearly demonstrates distinct regulation of a specific gene, COX-2, by a giving stimulus, IL-1ß, in ectopic vs. eutopic endometrial cells.

Supported by a growing body of evidence, the biochemical nature of ectopic endometriotic tissues, although emerging from the same origin, is different from its eutopic counterpart (2, 3, 4, 24, 25, 26). However, none of them was able to pinpoint exactly how this was achieved. To investigate the potential mechanism underlying distinct biochemical responsiveness in eutopic and ectopic endometria, we first evaluated the expression of COX-2 in response to IL-1ß because it is one of the primary inflammatory cytokines in peritoneal fluid of women with endometriosis. We demonstrated that the expression of COX-2 in response to an IL-1ß challenge in ectopic endometriotic stromal cells was much greater than that in eutopic endometrial stromal cells. This phenomenon was indisputably observed in unpaired normal and endometriotic samples as well as paired eutopic and ectopic samples collected from the same individual, indicating this is not just a random event due to different genetic backgrounds. Rather, it reflects that distinct mechanisms are used by IL-1ß to regulate COX-2 expression and PGE2 production in normal and endometriotic tissues.

There are several possibilities leading to increased sensitivity of COX-2 expression in response to IL-1ß in ectopic endometriotic stroma. First, there are more IL-1ß receptors in ectopic than eutopic endometrial stroma. This possibility can be ruled out because we demonstrated, by RT-PCR and Western blot analysis, that concentrations of mRNA and protein for specific IL-1ß receptor (IL-1R1) and/or decoy receptor (IL-1R2) are similar in eutopic and ectopic endometrial stroma. The second possibility is that different signaling pathways are used by eutopic and ectopic endometrial stromal cells due to yet unclear mechanisms. This is also unlikely as evident in that signaling molecules activated by IL-1ß in both cells are almost indistinguishable. Nevertheless, contributions from yet uncharacterized signaling molecules cannot be completely ruled out. The third possibility is that ectopic endometriotic stromal cells can produce IL-1ß, which binds to its cognate receptor via autocrine/paracrine manner to augment its COX-2-inducing capability. Although we did not measure the production of IL-1ß by eutopic and ectopic endometrial stromal cells in the current study, this is still an unfavorable scenario because it is has been reported that cultured stromal cells obtained from ectopic endometriotic tissue and its eutopic counterpart produced similar amounts of IL-1ß (27). The fourth possibility is that IL-1ß distinctly regulates COX-2 mRNA stability in eutopic and ectopic endometrial stroma. A recent study by Tamura et al. (28) showed that IL-1ß increased COX-2 mRNA stability in normal endometrial stromal cells, which is mediated via ERK1/2-dependent pathways. Others (29, 30) also reported that p38 MAPK is involved in COX-2 mRNA stabilization. Thus, it is possible that IL-1ß will have a distinct effect on COX-2 mRNA stabilization in eutopic vs. ectopic endometrial stroma. By showing that IL-1ß indistinguishably enhances the half-life of COX-2 mRNA in both eutopic and ectopic stromal cells, which can be blocked by administration of selective MEK, p38 MAPK, and IKK inhibitors, we also have to rule out this possibility.

The last but not least possibility is that IL-1ß distinctly regulates COX-2 gene activity at the transcriptional level. Our data demonstrated that, in agreement with those reported by Tamura et al. (28), COX-2 promoter is not activated by IL-1ß in eutopic endometrial stromal cells. Intriguingly, we found that IL-1ß significantly increases COX-2 promoter activity in ectopic endometriotic stromal cells in a dose-dependent manner. To rule out that IL-1ß-induced COX-2 promoter activity is due to a difference in genetic backgrounds of endometriosis-free and endometriosis patients, we repeated the transient transfection promoter assay in four sets of paired samples (one pair was not used because the ectopic stromal cell was resistant to transfection), and identical results were obtained. Thus, the distinct responsiveness of COX-2 gene to IL-1ß is likely mediated via differential regulation of COX-2 promoter.

Up-regulation of COX-2 promoter activity by IL-1ß has been reported in numerous studies. In sum, transcription factors including CREB, CCAAT/enhancer-binding protein, activator protein-1, and NF-{kappa}B were found to play important roles in regulating IL-1ß-induced COX-2 promoter activity (31, 32, 33, 34). Interestingly, all of these transcription factors bind to cis-elements located within 500 bp of the transcription start site. In this study, however, we found the CRE site located at –571/–564 is the most critical region for IL-1ß induced COX-2 promoter activity in ectopic endometriotic stromal cells. The discrepancy between our data and others is not known. Nevertheless, three lines of evidence clearly support our conclusion. First, IL-1ß fails to induce promoter activity in constructs containing –550/+49 or shorter, which lack the functional CRE; second, site-directed mutagenesis of the CRE located at –571/–564 abrogates IL-1ß-induced COX-2 promoter activity; and third, EMSA data (direct binding and specific competition) demonstrated that CREB directly binds to the CRE of this region.

It is known that CREB can be phosphorylated by the ERK or p38 MAPK-dependent pathway (22, 35) and that phosphorylation of CREB can recruit a transcription coactivator such as CREB binding protein or P300, resulting in activation of basic transcriptional machinery. In this study, we demonstrate that IL-1ß induces CREB phosphorylation and U0126 and SB202190 inhibit this effect, indicating IL-1ß-induced CREB phosphorylation is indeed mediated via the ERK- and p38 MAPK-dependent pathway. Moreover, treatment with an MEK inhibitor completely abrogates IL-1ß-induced COX-2 promoter activity, whereas a p38 MAPK inhibitor only partially decreased IL-1ß-driven COX-2 promoter activity. This result is nicely mirrored in that U0126 inhibits IL-1ß-induced COX-2 protein expression more effectively than that by BAY and SB in ectopic endometriotic stromal cell but not in eutopic endometrial stroma. The data can be explained by the fact that only the ectopic endometriotic stromal cell exerts IL-1ß-induced COX-2 promoter activity, whereas eutopic endometrial stroma does not possess such a capability. Therefore, NF-{kappa}B signaling cascades contribute only to enhancement of mRNA stability, whereas ERK and p38 MAPK pathways can increase not only COX-2 promoter activity but also mRNA stability.

In summary, IL-1ß increased COX-2 expression and concomitant PGE2 production in both eutopic and ectopic endometrial stromal cells. The induction of the COX-2 expression and PG secretion by IL-1ß is through transcriptional regulation of COX-2 gene via activation of the ERK/p38 MAPK-dependent CREB phosphorylation in ectopic endometrial stromal cells and posttranscriptional enhancement of COX-2 mRNA stability. Increased sensitivity of IL-1ß-dependent COX-2 expression in endometriotic stromal cells may play a critical role in the pathophysiology of the endometriosis development.


   Acknowledgments
 
The authors are grateful for the valuable assistance of COX-2 promoter analysis by Bioinformatics Center at the National Cheng Kung University.


   Footnotes
 
This work was supported by grants from the National Science Council of Republic of China (NSC92-2320-B006-080 and NSC91-2314-B-006-126) and in part by a grant from the National Cheng Kung University Hospital (NCKUH 92-076).

First Published Online October 13, 2004

1 M.-H.W. and C.-A.W. contributed equally to this work. Back

Abbreviations: COX, Cyclooxygenase; CRE, cAMP response element; CREB, cAMP response element-binding protein; EIA, enzyme immunoassay; IKK, I{kappa}B kinase; JNK, c-Jun N-terminal kinase; MEK, MAPK kinase; NF-{kappa}B, nuclear factor-{kappa}B; PG, prostaglandin.

Received August 12, 2004.

Accepted October 7, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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