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Departments of Obstetrics and Gynecology and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60612
Address all correspondence and requests for reprints to: Serdar E. Bulun, M.D., Departments of Obstetrics and Gynecology and Molecular Genetics, University of Illinois at Chicago, 820 South Wood Street, M/C 808, Chicago, Illinois 60612. E-mail: . sbulun{at}uic.edu
Abstract
We investigated the regulation of PG production in human endometrial stromal cells (ESC) by IL-1ß. We found that cyclooxygenase-2 (COX-2) mRNA and protein levels and PGE2 production in ESC were significantly increased by IL-1ß. COX-2 mRNA, protein, and PGE2 levels in IL-1ß-treated ESC were decreased by a PKA inhibitor, a nuclear factor (NF-
B) inhibitor, and an ERK1/2 inhibitor, but not by a p38 MAPK inhibitor or a PKC inhibitor, suggesting the possible involvement of PKA, NF-B, and/or the ERK1/2 signaling pathway(s) in IL-1ß-mediated COX-2 gene induction in ESC. We then transiently transfected deletion mutants of the COX-2 promoter fused to the luciferase reporter gene and variants of -360/+56 bp promoter construct carrying different site-directed mutations of selected cis-acting elements. We determined that a NF-B site (-222/-213 bp), a nuclear factor for IL-6 expression site (NF-IL6, -132/-124 bp), and a cAMP response element (-59/-52 bp) were essential for the baseline COX-2 gene promoter regulation. The addition of IL-1ß, however, did not affect the activity of these COX-2 promoter constructs. To investigate the potential effects of IL-1ß on COX-2 mRNA stability, ESC were treated with actinomycin D, a general transcription inhibitor, in the absence or presence of IL-1ß. We found that 1) IL-1ß significantly increased COX-2 mRNA stability; 2) continuous transcription was not required to sustain the IL-1ß-induced COX-2 mRNA levels; and 3) COX-2 mRNA was highly unstable in the absence of IL-1ß. Additionally, we found that the ERK1/2 signaling pathway was essential for stabilizing COX-2 mRNA. We conclude that levels of COX-2 mRNA, protein, and enzyme activity in ESC are controlled by various signaling pathways, including PKA, ERK1/2, and NF-B. Moreover, posttranscriptional mRNA stability is an important mechanism for IL-1ß-induced elevation of COX-2 expression in ESC.
SUPPORTED BY A plethora of experimental evidence, PG production emerged as a highly promising therapeutic target in the treatment of many inflammatory diseases. The increased amounts of PGs in inflammatory diseases reflect enhanced synthesis, which occurs by cyclooxygenase (COX)-catalyzed metabolism of arachidonic acid. PGs are synthesized from arachidonic acid by two different isoforms of COX, referred to as COX-1 and COX-2. They share over 60% identity at the amino acid level and have similar enzymatic activities, but although they catalyze the same reaction, the isoforms may have distinct biological functions (1, 2, 3, 4). COX-1 is constitutively expressed in most mammalian tissues and is thought to carry out housekeeping functions, such as cytoprotection of the gastric mucosa, regulation of renal blood flow, and control of platelet aggregation. In contrast, COX-2 mRNA and protein are normally undetectable in most tissues, but can be rapidly induced in response to proinflammatory or mitogenic stimuli, including various cytokines, growth factors, oncogenes, endotoxins, and chemicals (5, 6, 7, 8, 9, 10).
Endometriosis, a common disease among women of reproductive age, is characterized by the presence and growth of endometrial tissue (glands and stroma) outside the uterus. Despite a long history of clinical experience and experimental research, endometriosis remains an enigma, and its pathogenesis is still controversial. Inflammation and chronic pain are two major features of endometriosis, which led the investigators to study PG production in this disease. Levels of PGs (especially PGE2) in endometriosis tissue had been shown to be higher than levels in normal endometrium (11, 12, 13, 14, 15). These results suggest that increased production of PGs in endometriosis is a result of enhanced COX-2 gene expression. An important general concept is that endometriosis is a local pelvic inflammatory process with altered function of immune-related cells in the peritoneal environment. Supporting this concept are recent studies suggesting that the peritoneal fluid of women with endometriosis contains an increased number of activated macrophages that secrete various local products, such as growth factors and cytokines (16, 17, 18, 19). Studies have reported elevated levels of several cytokines in the peritoneal fluid of women with endometriosis, thus implicating these cytokines in the development and progression of endometriosis. IL-1ß, one of those cytokines, was shown to increase COX-2 gene expression in several kinds of cells (5, 6, 7). It has also been implicated in endometrial and endometriotic function (20, 21, 22).
Therefore, we hypothesized that endometriotic cells and/or immune cells, especially macrophages, secrete IL-1ß, which acts in an autocrine and/or paracrine fashion to increase COX-2 expression in the endometriosis and endometrium. Bergqvist et al. (20) showed that endometriotic tissue itself had significantly higher concentrations of IL-1ß and IL-6 than normal endometrium. Therefore, to determine the direct effect of IL-1ß on COX-2 expression, we used normal human endometrial stromal cells (ESC) as a model system to test this hypothesis. In addition, we attempted to identify the signal transduction pathway(s) involved and characterize the critical cis-acting elements that mediate induction the human COX-2 gene in ESC.
Materials and Methods
Reagents
U0126 (specific inhibitor of ERK1/2) and SB203580 (specific inhibitor of p38 MAPK) were purchased from Promega Corp. (Madison, WI). IL-1ß, indomethacin (nonselective COX-1 and -2 inhibitor), NS-398 (selective COX-2 inhibitor), sodium salicylate (NaS), actinomycin D (Act D; general transcription inhibitor), 1-pyrrolidinecarbodithioic acid [PDTC; a nuclear factor-B (NF-B) inhibitor], PKA inhibitor (PKAI) amide fragment 6–22, and PKC inhibitor (PKCI) amide fragment 19–36 were purchased from Sigma-Aldrich Corp. (St. Louis, MO). All other materials used in the study are indicated in appropriate context below.
Cell culture
Human normal endometrial tissues were obtained at the time of surgery from reproductive aged women (n = 12) who were undergoing hysterectomy for advanced cervical dysplasia after obtaining informed consent following a protocol approved by the Office for Protection of Research Subjects of the University of Illinois (Chicago, IL). These patients did not receive hormonal treatments or take antiinflammatory drugs (especially COX inhibitors) before surgery. Six specimens were from the proliferative phase; the other six were from the secretory phase. No differences in experimental results were noted with respect to cycle phase. Normal human ESC were cultured using a previously reported protocol (23). Briefly, endometrial tissues were minced finely and digested with collagenase B (1 mg/ml) at 37 C for 2 h. Single cell suspensions were prepared by filtration through a 75-µm pore size sieve. Fresh cells were suspended in DMEM/Ham’s F-12 containing 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin (250 ng/ml; growth medium) in a humidified atmosphere with 5% CO2 at 37 C. Twelve to 24 h after attachment of ESC, culture medium was changed at 48-h intervals until the cells became confluent. Confluent ESC were serum-deprived for 16 h in serum-free, phenol red-free DMEM/F-12 before being subjected to the following treatments: 1) serum-free, phenol red-free DMEM/F-12 as the baseline control; and 2) serum-free, phenol red-free DMEM/F-12 with IL-1ß. ESC incubated under these conditions were then used to isolate total RNA for RT-PCR; whole cell protein extracts for Western blot analysis.
Semiquantitative RT-PCR amplification
ESC were cultured in 100-mm dishes until confluent in the growth medium as described above, then were switched to serum-free, phenol red-free medium for 16 h. These cells were incubated under various conditions, i.e. control, IL-1ß (1 ng/ml), IL-1ß (1 ng/ml) with a signal transduction inhibitor, or IL-1ß (1 ng/ml) with NaS for 2, 4, or 8 h. Total RNA was isolated from ESC using the RNeasy mini kit (QIAGEN, Valencia, CA) following the protocol suggested by the manufacturer. The integrity of the RNA was confirmed by agarose gel electrophoresis. For RT-PCR analysis of COX-2 mRNA, the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) was used to synthesize the first strand cDNA as instructed by the supplier. Briefly, 5 µg total RNA isolated from ESC were treated with deoxyribonuclease I (DNase I; 1 U/µl). One microliter of this was reserved for PCR amplification with primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing a control for equal starting amounts of total RNA in samples and PCR efficiency. The remainder of the DNase-treated RNA was directly reverse transcribed. One microliter of the reverse transcriptase reaction mix was used for PCR with oligonucleotide pairs specific for COX-2 and GAPDH. For COX-2, the PCR primers included the +573/+878 bp coding region, and the expected size of the PCR product is 305 bp. For GAPDH, the primers included the coding region from +216 to +593 bp, with an expected product size of 593 bp. The nucleotide sequences of the primer pairs employed were as follows (24): for COX-2, 5'-TTCAAATGAGATTGTGGGAAAAT-3' and 5'-AGATCATCTCTGCCTGAGTATCTT-3'; and for GAPDH, 5'-CCACCCATGGCAAATTCCATGGCA-3' and 5'-TCTAGACGGCAGGTCAGGTCCACC-3'. For COX-2, amplification was obtained with 1 cycle of 95 C for 2 min, followed by 30 cycles of 95 C for 1 min, 58 C for 2 min, and 72 C for 1 min. For GAPDH, PCR conditions were the same, except that the annealing temperature was 60 C. PCR performed with the original RNA sample after DNase I digestion (see above) did not yield any product, confirming that amplified products were dependent on the presence of template generated by RT and were not the result of contamination with extraneous DNA. Aliquots of the reaction products were analyzed by electrophoresis in an agarose gel and with ethidium bromide staining. The intensity of the PCR products was quantified using Quantity One 1-D Analysis software (Bio-Rad Laboratories, Inc., Hercules, CA). We assert that these data are semiquantitative (relative to control GAPDH) based on the following test performed before data analysis. Both products were assayed in the linear response range of the RT-PCR amplification process; the cycle number used in this assay was determined by finding the midpoint of linear amplification on a sigmoidal curve for both amplification products, with cycle numbers 25–35 plotted against band density (see Fig. 1A
).
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ESC were cultured in 100-mm dishes until confluent in the growth medium as described above and were switched to serum-free, phenol red-free medium for 16 h. These cells were then incubated under various conditions, i.e. control, IL-1ß (1 ng/ml), or IL-1ß (1 ng/ml) with a signal transduction inhibitor for 4, 8, or 16 h. Total protein was extracted from whole cells using M-PER Mammalian Protein Extraction Reagent (Pierce Chemical Co., Rockford, IL) following the protocol suggested by the manufacturer. The protein concentration was measured with a bicinchoninic acid protein assay kit (Pierce Chemical Co.), according to the manufacturer’s instructions. The lysate (20 µg total protein) was mixed with 6 x standard electrophoresis sample buffer and fractionated by SDS-PAGE (4% stacking gel, 7.5% resolving gel) at 25 mA for 4 h. Protein samples were then electroblotted to the Transblot nitrocellulose membrane (0.2 µm pore size; Bio-Rad Laboratories, Inc.). The membrane was incubated with an anti-COX-2 polyclonal antibody at a 1:5,000 dilution (0.04 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in blocking buffer for 1 h at room temperature and then incubated similarly with horseradish peroxidase-conjugated antigoat IgG (Santa Cruz Biotechnology, Inc.), diluted 1:100,000 in blocking buffer, for 1 h at room temperature. The signal was detected using the SuperSignal West Femto Maximum Sensitivity Substrate Chemiluminescence Kit (Pierce Chemical Co.) according to the manufacturer’s protocol and was exposed to Biomax ML x-ray film (Kodak, Rochester, NY) for less than 2 min. The band intensity of protein expression was quantified using Molecular Analyst software (version 1.5, Bio-Rad Laboratories, Inc.).
PGE2 measurements
ESC were plated in six-well tissue culture plates in the growth medium as described above and allowed to become established as confluent monolayers for 24 h. All cells were serum-depleted for at least 16 h and not treated (control) or treated with IL-1ß (1 ng/ml), IL-1ß (1 ng/ml) with a COX enzyme inhibitor, or IL-1ß (1 ng/ml) with a signal transduction inhibitor (total volume, 2 ml/well). Stimulations were performed for 24 h, and the supernatants were transferred to clean microcentrifuge tubes. Two 100-µl aliquots of supernatant/sample were assayed using a PGE2 immunoassay kit (R\|[amp ]\|D Systems, Inc., Minneapolis, MN), according to the manufacturer’s instructions. The concentration of PGE2 was determined for competitive binding ELISA using a Microplate Reader (model 550, Bio-Rad Laboratories, Inc.). These measurements were made in duplicate and repeated in three separate experiments.
Plasmid construction
Construction of the deletion mutants containing specific regions of the human COX-2 gene promoter in the luciferase reporter vector pGL3 Basic (Promega Corp.) was accomplished by PCR amplification of the desired region using the recombinant plasmid containing a 7-kb promoter region of the human COX-2 gene (provided Dr. Stephen M. Prescott, University of Utah, Salt Lake City, UT) as the template. For each PCR, primer pairs used to amplify specific regions of the COX-2 promoter region had suitable flanking restriction sites that forced cloning of the fragment in the desired orientation into the pGL3 Basic vector. The orientation and sequence of all constructs were verified by direct sequencing using the ABI PRISM 377 DNA sequencer (PE Applied Biosystems, Foster City, CA).
Site-directed mutagenesis
Mutant constructs, phCOX2(CRM), with a mutation at the cAMP response element (CRE); phCOX2(ILM), with a mutation at the putative nuclear factor for IL6 expression (NF-IL6) site; and phCOX2(KBM), with a mutation at the NF-B site, were constructed as described previously (25, 26). Briefly, for the CRE, TTCGTCA was changed to TTgagCt; for the NF-IL6 site, the sequence was changed from TTACGCAAT to TTggtaccT; and, for the NF-B site, the sequence was changed from GGGGACTACC to GGccACTACC; the lowercase nucleotides indicate the mutations. The mutations and the orientation of insert were confirmed by direct sequencing. Plasmids used in transfection experiments were purified using an EndoFree Plasmid Isolation Kit (QIAGEN, Valencia, CA), and purity was verified by spectrophotometry and agarose gel electrophoresis.
Transient transfections and luciferase assays
The day before transfection, ESC were plated into six-well tissue culture plates at a density such that the cells reached 70–80% confluence by the time of transfection. Transfections were performed using with Lipofectamine Plus reagent (Invitrogen), following the protocol provided by the manufacturer. Each transfection was performed using 0.4 µg firefly luciferase reporter construct DNA that contained serial deletion and site-specific mutants of COX-2 promotor gene and 1 ng of an internal control Renilla luciferase reporter plasmid pRL-TK (Promega Corp.). Three hours after transfection, the transfection medium was removed by aspiration, DMEM/F-12 (2 ml) containing 10% FBS and antibiotics was added, and the plates were returned to the incubator for 16 h. Cells received serum-free DMEM/F-12 for an additional 16 h and were then switched to control or IL-1ß (1 ng/ml) for another 24 h. Then, medium was removed, and wells were rinsed with PBS to remove detached cells and residual growth medium. Two hundred and fifty microliters of 1 x passive lysis buffer, provided in the Dual-Luciferase Reporter Assay System (Promega Corp.), were added per well. Ten microliters of supernatant were used for the assay of luciferase activities. Luciferase activities were determined using the LUMAT LB9507 luminometer (Berthold Technologies GmbH & Co.KG, Bad Wildbad, Germany). Firefly luciferase activities were normalized based on the Renilla luciferase activity in each well. These measurements were performed in triplicate and repeated in three independent experiments.
Statistical analysis and data expression
Statistical analysis for the comparison of COX-2 expression or PGE2 production was performed with an unpaired t test using the StatView 5.0 statistical software package (SAS Institute, Inc., Cary, NC). P < 0.05 was considered significant. All values are given as the mean, with the bars (in all figures) showing the SEM.
Results
Effects of IL-1ß on COX-2 mRNA and protein levels and PGE2 production in normal ESC
We initially carried out experiments to evaluate the optimal conditions for determining the effects of IL-1ß on COX-2 mRNA levels in ESC. Treatment of ESC with IL-1ß (0.01, 0.1, 1, and 10 ng/ml) gave rise to dose-dependent increases in COX-2 mRNA levels determined by semiquantitative RT-PCR. COX-2 mRNA levels in ESC were strikingly induced after IL-1ß treatment at 0.01 ng/ml and reached a plateau at 1 ng/ml. Single PCR products were obtained for COX-2 (data not shown). Based on this observation, ESC were treated with 1 ng/ml IL-1ß. To determine where PCR amplification for COX-2 mRNA was in the logarithmic phase, total RNA isolated from ESC treated with or without IL-1ß was reverse transcribed and was amplified under different cycle numbers (Fig. 1A
). A linear relationship between PCR products and amplification cycles was observed for COX-2 in ESC in the presence of IL-1ß. However, we could detect PCR yields of COX-2 mRNA as a faint band in just 40 cycles in untreated control cells. Consequently, 30 cycles of PCR were employed for quantification of IL-1ß-induced.
We next investigated the time dependency of the induction of COX-2 mRNA and protein synthesis by IL-1ß. Compared with the control, IL-1ß treatment significantly increased COX-2 mRNA levels in ESC (Fig. 1B). COX-2 mRNA levels in ESC were strikingly induced after IL-1ß treatment at 2 h and reached a maximum at 4 h, whereas COX-2 mRNA was not detected in control cells. PCR was also performed using an aliquot of the RT products for the housekeeping gene GAPDH mRNA to control the RT reaction, PCR efficiency, and equal starting amounts of total RNA. There was no apparent change in GAPDH mRNA abundance upon IL-1ß treatment. COX-2 protein levels were also induced after IL-1ß treatment at 4 h and remained detectable up to 16 h (Fig. 1C). Equal loading of protein in each lane was confirmed by Coomassie blue staining of samples fractionated on SDS-PAGE. Quantitative densitometry for three independent experiments confirmed these results.
To determine whether the induction of COX-2 mRNA and protein levels was correlated with comparable changes in PGE2 production, PGE2 concentrations were measured in culture medium of ESC after IL-1ß (1 ng/ml) treatment. The effect of IL-1ß on PGE2 synthesis in ESC is shown in Fig. 1D. Incubation of ESC with IL-1ß for 24 h caused a significant increase in the PGE2 concentration in the medium by 2.1-fold compared with the medium from untreated control cells. Additionally, pretreatment with 50 µM indomethacin (nonselective COX-1 and -2 inhibitor) or 50 µM NS-398 (selective COX-2 inhibitor) for 30 min significantly decreased PGE2 synthesis in ESC incubated with IL-1ß.
Levels of COX-2 mRNA, protein, and PGE2 production in IL-1ß-treated ESC are decreased by inhibitors of PKA, NF-B and ERK1/2 signal transduction pathways
After establishing that IL-1ß can induce COX-2 expression in ESC, we studied the signaling pathway(s) mediating the action of IL-1ß. ESC were pretreated with various inhibitors of signal transduction for 30 min and then were incubated with IL-1ß (1 ng/ml) for 4 h. PKAI (50 nM; IC50, 1.7 nM), 1 µM U0126 (ERK1/2 inhibitor; IC50, 0.53 µM), and 20 µM PDTC (NF-B inhibitor; IC50, 15 µM) caused significant decreases in the density of the COX-2 mRNA band (27, 28, 29) (Fig. 2A). Recent reports of the ability of NaS to suppress not only COX enzyme activity, and the IL-1ß-mediated COX-2 gene induction prompted us to examine whether NaS was also capable of inhibiting IL-1ß-mediated induction of the COX-2 gene expression in ESC (30). NaS was shown to prevent NF-B activation and affect the ERK1/2 signaling pathway (31, 32). As shown in the Fig. 2B, preincubation of ESC for 30 min with a therapeutically relevant concentration (100 µM) of NaS before the addition of IL-1ß markedly reduced the intensity of the COX-2 mRNA band, as evaluated by RT-PCR. COX-2 immunoblot experiments confirmed that U0126, PKAI, and PDTC indeed abolished the increased COX-2 protein level otherwise seen upon IL-1ß treatment (Fig. 2C). Equal loading of protein in each lane was confirmed by Coomassie blue staining of samples fractionated on SDS-PAGE. These inhibitors also decreased PGE2 production in IL-1ß-treated ESC (Fig. 2D). In contrast, pretreatment with several concentrations of PKCI (IC50, 15 µM) or SB203580 (p38 MAPK inhibitor; IC50, 0.6 µM) did not affect COX-2 mRNA and PGE2 synthesis in IL-1ß-treated ESC (33, 34) (representative data shown in Fig. 2, A and D). These results suggested that IL-1ß-induced COX-2 expression in ESC may be mediated via PKA, NF-B, and/or activation of ERK1/2 and not via PKC or the p38 MAPK pathway.
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Deletion mutants of COX-2 promoter-driven luciferase reporter gene constructs were transiently transfected to ESC and treated with or without IL-1ß (1 ng/ml) for 24 h (Fig. 3A). The -360/+56 bp region gave rise to the highest level of COX-2 promoter activity. IL-1ß did not affect the transcription of any COX-2 reporter construct. Sequence analysis of this region revealed existence of a CRE at -59/-52 bp, a NF-IL6 site at -132/-124 bp, and a NF-B site at -222/-213 bp. Site-directed mutations of the CRE, NF-IL6 site, and the NF-B site either alone or in combination significantly reduced COX-2 promoter activity in ESC (Fig. 3B). These results suggested that CRE, NF-IL6, and NF-B sites were critical for COX-2 promoter activity, and that IL-1ß did not alter the promoter activity of these site-directed mutant constructs.
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Based on the data from transfection studies obtained in the current study (Fig. 3), it is unlikely that IL-1ß modifies COX-2 expression in ESC at the transcriptional level. Hence, we examined the role of a posttranscriptional mechanism(s) involving mRNA stabilization. To address this possibility, we employed a classical technique involving the measurement of COX-2 mRNA levels in transcriptionally arrested cells using Act D in the absence or presence of IL-1ß (Fig. 4). First, ESC in culture were treated with IL-1ß (1 ng/ml) for 4 h (maximum mRNA level; see Fig. 1B). At this time point (control, 0 h) we distinguished four conditions. In the first two conditions the medium already containing IL-1ß was retained for another 4 h [Fig. 4; •, control cells with no additions; 
, addition of the transcriptional inhibitor Act D (10 µg/ml) to unchanged medium (with IL-1ß)]. Total RNA was isolated at various time points after the additional treatments and was examined for the presence of COX-2 mRNA by semiquantitative RT-PCR. For correction for differences in loading, the signal density of each COX-2 band was divided by the signal density of the GAPDH band. No difference in mRNA levels between these two conditions was observed, indicating that mRNA stability induced by IL-1ß was the major mechanism responsible for the steady state levels. On the other hand, when IL-1ß was removed by replacing the original medium with IL-1ß-free fresh medium, steady state mRNA levels declined significantly (Fig. 4, 
and 
). The absence or presence of Act D did not cause a difference. This experiment was performed on three different occasions with reproducible results. These results suggest that 1) IL-1ß significantly increased COX-2 mRNA stability; 2) continuous transcription is not required to sustain the IL-1ß-induced COX-2 mRNA levels; 3) COX-2 mRNA is highly unstable in the absence of IL-1ß (t1/2 = 3.0 h); and 4) Act D has no effect on the rate of COX-2 mRNA decay. Therefore, the stabilization of COX-2 mRNA may be the major mechanism for IL-1ß-induced elevation of COX-2 expression in ESC.
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A similar approach in previous experiments was initiated to evaluate the effect of PKAI, U0126 (ERK1/2 inhibitor), or PDTC (NF-B inhibitor) on the stability of IL-1ß-induced COX-2 mRNA. First, ESC in culture were treated with IL-1ß (1 ng/ml) for 4 h (maximum mRNA level; see Fig. 1B). At this time point (control, 0 h) we distinguished four conditions. The first two conditions were the same as those in previous experiments; that is, the medium already containing IL-1ß was retained for another 4 h (Fig. 5, A–C; •, control cells with no additions; , addition of 10 µg/ml Act D, the transcriptional inhibitor, to the medium with IL-1ß). In the latter two conditions, either Act D or Act D plus a signal transduction inhibitor (PKAI, U0126, or PDTC) was added (Fig. 5, A–C, and ). Total RNA was isolated at various time points after the additional treatments and was examined for the presence of COX-2 mRNA by semiquantitative RT-PCR. For correction for differences in loading, the signal density of each COX-2 band was divided by the signal density of the GAPDH band. The addition of U0126 (1 µM) together with Act D to the cells after 4 h of stimulation with IL-1ß resulted in a rapid decrease (t1/2 = 3.5 h) in COX-2 mRNA levels (Fig. 5B). When U0126 alone was added to cells treated with IL-1ß for 4 h, there was also a rapid decay of COX-2 mRNA that occurred at a similar rate as in cells treated with U0126 plus Act D. On the other hand, little or no significant difference in mRNA decay was observed after the addition of PKAI (50 nM) or PDTC (20 µM) with or without Act D (Fig. 5, A and C). These experiments were performed on three different occasions with reproducible results. These results suggest that posttranscriptional events are involved in the inhibition of IL-1ß-induced COX-2 expression by U0126, indicating that the ERK1/2 signaling pathway is essential for stabilizing COX-2 mRNA. In contrast, PKAI and PDTC do not affect the stability of COX-2 mRNA.
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In the present study we have demonstrated the role of IL-1ß in up-regulating the expression of COX-2 at the mRNA, protein, and enzyme activity levels in human ESC as a model system of inflammatory diseases. We also found that COX-2 gene induction by IL-1ß involved the ERK1/2 signaling pathway. Since the classic work of Vane (35), it has been widely accepted that the pharmacological action of nonsteroidal antiinflammatory drugs (NSAID), i.e. indomethacin, NS-398, and aspirin, is mediated primarily by inhibiting the activity of COX enzyme. We have shown that both indomethacin (nonselective COX-1 and -2 inhibitor) and NS-398 (selective COX-2 inhibitor) inhibit COX-2 enzyme activity. They reportedly bind to the enzyme active site and subsequently cause a slow, irreversible event that leads to significant inactivation of the enzyme (36). Therefore, these inhibitors blocked PGE2 production, but had no effect on COX-2 mRNA or protein (37). On the other hand, aspirin (acetylsalicylic acid), a nonselective COX inhibitor, is rapidly deacetylated in blood to form salicylic acid (38, 39). NaS, a derivative of salicylic acid, has been a known NSAID for over a century and administered in the treatment of many diseases, but its mechanism of action remains a pharmacological enigma. The inhibitory effect of NaS on COX-2 expression appears selective and is not shared by indomethacin or NS-398. Recently, NaS was shown to prevent NF-B activation in human Jurkat T cells and murine pre-B cells by preventing the proteolytic degradation of IB (31). These studies have been extended to demonstrate that NaS, but not other NSAID, such as indomethacin, inhibits endothelial and monocytic gene expression by preventing the phosphorylation and degradation of IB (40, 41). Schwenger et al. (32) demonstrated that NaS also affected the ERK1/2 signaling pathway. Intriguingly, Saunders et al. (30) recently demonstrated that NaS at a therapeutic concentration suppressed COX-2 gene transcription induced by IL-1ß by inhibiting the binding of CCAAT/enhancer-binding protein-ß to the NF-IL6 site. We have shown here that NaS at therapeutic concentrations suppresses COX-2 gene transcription in ESC. We speculate that the suppression of IL-1ß-induced COX-2 expression by NaS in ESC is mediated through one or more signal transduction pathway.
U0126 is an organic compound [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] that has been identified as an inhibitor of activating protein-1 trans-activation in cell-based reporter assays (28). It specifically inhibits the phosphorylation and activation of ERK1/2. U0126 does not affect the phosphorylation of MAPK kinase (MEK), the upstream kinase of ERK, at concentrations sufficient to inhibit ERK phosphorylation. It thus appears that U0126 inhibits MEK directly by inhibiting the catalytic activity of the active enzyme. In addition, U0126 shows little, if any, effect on the kinase activities of PKC, Abl, Raf, MEKK, c-Jun N-terminal kinase, or cyclin-dependent kinase-2 or -4. Recent studies have suggested that Ras (the upstream kinase of MEK) activation may induce COX-2 expression in several systems. Activated, oncogenic H-Ras was inducibly expressed in Rat-1 fibroblasts with a concomitant increase in COX-2 expression as well as PGE2 production (42) In this particular cell line a specific ERK inhibitor was found to suppress COX-2 induction by oncogenic Ras, suggesting that ERK1/2 activation is required for Ras-dependent induction of COX-2. Similarly, elevated levels of COX-2 as well as PGE2 were detected in Ras-transformed mammary epithelial cells (43). On the other hand, PDTC, a potent inhibitor of NF-B, reversibly suppresses the release of an inhibitory subunit IB from the latent cytoplasmic form of NF-B in cells (44, 45). PDTC is known to possess at least two chemical properties. One is potent antioxidative activity; the second is a heavy metal chelating activity. These two properties are believed to participate in the inhibitory mechanism of PDTC, but the details are not known. Therefore, the suppression of COX-2 expression by U0126 or PDTC is in agreement with our observation that ERK1/2 and NF-B are important in the regulation of COX-2 in ESC.
In the present study we determined that CRE at -59/-52, the NF-IL6 site at -132/-124, and the NF-B site at -222/-213 in the COX-2 promoter gene were critical for the optimal COX-2 transcriptional increase in ESC. However, as the reporter construct containing the COX-2 promoter region -360/+56 produced the maximum stimulation, and we did not employ additional experiments to corroborate this observation, we do not rule out the possibility of cooperative involvement of the other sites to elicit COX-2 promoter in ESC. Interestingly, IL-1ß did not affect the transcription of any of the COX-2 reporter constructs. Previous investigators reported similar results of a dissociation of COX-2 mRNA levels and promoter activity in ESC (7). Recent studies by Gilroy et al. (46) demonstrated that COX-2 expression was regulated in a cell cycle-dependent manner. Cells in the G0 state were highly responsive to COX-2 induction by exogenous stimuli, whereas COX-2 expression in cells entering the growth phase progressively declined. Even though we did not specifically examine this in the current study, it is possible that the cell cycle phase of ESC affected the COX-2 promoter activity induced by IL-1ß.
It now appears from published evidence that the COX-2 gene is regulated through both 5' (transcriptional) and 3' (posttranscriptional) regulatory elements after IL-1ß signal activation (47, 48, 49, 50). Early studies by Raz et al. (51) demonstrated that inducible COX-2 synthesis could be divided into early transcriptional and late posttranscriptional phases. The kinetics of transcriptional activation (1–3 h) of the COX-2 gene could not account for the sustained kinetics of induction demonstrated by IL-1ß (52). The half-life of COX-2 mRNA was approximately 3 h in the present study. High levels of encoded protein products from COX-2 genes are usually required for only a short period of time and must be expressed in a burst (50). The entire 3'-untranslated region (2.5 kb) of the human COX-2 gene is encoded by exon 10, which contains three canonical (AAUAAA) polyadenylation sequences and 22 copies of AUUUA Shaw-Kamen motifs (47, 48, 49, 50). The latter sequences are believed to be associated with message instability, translational efficiency, and rapid turnover (53, 54, 55). As COX-2 mRNA is highly unstable, and IL-1ß stabilizes COX-2 mRNA in the absence of transcription, we suggest that posttranscriptional mRNA stability is an important consequence of IL-1ß action. We also found that the inhibition of ERK1/2 activity significantly reduced the stability of the COX-2 mRNA (Fig. 5B). It is likely that the ERK1/2 pathway influences the proteins that bind to the AUUUA sequence of COX-2 mRNA, resulting in mRNA stabilization. Indeed, several molecules, such as AU-rich element RNA-binding protein and adenosine-uridine binding factor, bind AU-rich sequences to reduce and increase mRNA stability, respectively (56, 57, 58). In addition, the formation of the AUUUA sequence-binding complexes and the accelerated mRNA turnover are dependent on phosphorylation (58).
The molecular mechanisms responsible for IL-1ß-dependent regulation of COX-2 expression promote new insights into the pathophysiology of inflammation and may lead to new therapeutic strategies capable of interrupting the inflammatory cascade at key points. Other investigators suggested that the regulation of COX-2 expression and function by IL-1ß in ESC was relevant to uterine biology (59, 60, 61). During pregnancy, uterine PG synthesis is regulated by cytokines such as IL-6, TNF
, and IL-1ß through COX-2 expression. These cytokines are produced locally in normal uterine tissues during pregnancy, namely from the implantation period until parturition. Recent evidence indicates that the COX-2-deficient mouse has defective decidualization and implantation (62). These findings suggest an important role for COX-2 not only in inflammation, but also in reproductive biology.
Acknowledgments
We thank Dr. Stephen M. Prescott for providing the COX-2 promoter plasmid.
Footnotes
This work was supported by NIH Grant HD38691 (to S.E.B.) and a fellowship award from the Japan Menopause Society, Tokyo, Japan (to M.T.).
Abbreviations: Act D, Actinomycin D; COX, cyclooxygenase; CRE, cAMP response element; DNase I, deoxyribonuclease I; ESC, human endometrial stromal cells; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEK, MAPK kinase; MEKK, MAPK kinase kinase; NaS, sodium salicylate; NF-B, nuclear factor-B; NSAID, nonsteroidal antiinflammatory drugs; PDTC, 1-pyrrolidinecarbodithioic acid; PKAI, PKA inhibitor; PKCI, PKC inhibitor.
Received December 12, 2001.
Accepted March 7, 2002.
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