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Molecular Endocrinology, doi:10.1210/me.2006-0112
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Molecular Endocrinology 20 (11): 2724-2733
Copyright © 2006 by The Endocrine Society

Progesterone Receptor Plays a Major Antiinflammatory Role in Human Myometrial Cells by Antagonism of Nuclear Factor-{kappa}B Activation of Cyclooxygenase 2 Expression

Daniel B. Hardy, Bethany A. Janowski, David R. Corey and Carole R. Mendelson

Departments of Biochemistry (D.B.H., D.R.C., C.R.M.), Obstetrics and Gynecology (C.R.M.), and Pharmacology (B.A.J., D.R.C.), North Texas March of Dimes Birth Defects Center, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038

Address all correspondence to: Carole R. Mendelson, Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9038, E-mail: carole.mendelson{at}utsouthwestern.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Spontaneous labor in women and in other mammals is likely mediated by a concerted series of biochemical events that negatively impact the ability of the progesterone receptor (PR) to regulate target genes that maintain myometrial quiescence. In the present study, we tested the hypothesis that progesterone/PR inhibits uterine contractility by blocking nuclear factor {kappa}B (NF-{kappa}B) activation and induction of cyclooxygenase-2 (COX-2), a contractile gene that is up-regulated in labor. To uncover mechanisms for regulation of uterine COX-2, immortalized human fundal myometrial cells were treated with IL-1ß ± progesterone. IL-1ß alone caused a marked up-regulation of COX-2 mRNA, whereas treatment with progesterone suppressed this induction. This was also observed in human breast cancer (T47D) cells. In both cell lines, this inhibitory effect of progesterone was blocked by RU486. Using chromatin immunoprecipitation, we observed that IL-1ß stimulated recruitment of NF-{kappa}B p65 to both proximal and distal NF-{kappa}B elements of the COX-2 promoter; these effects were diminished by coincubation with progesterone. The ability of progesterone to inhibit COX-2 expression in myometrial cells was associated with rapid induction of mRNA and protein levels of inhibitor of {kappa}B{alpha}, a protein that blocks NF-{kappa}B transactivation. Furthermore, small interfering RNA-mediated ablation of both PR-A and PR-B isoforms in T47D cells greatly enhanced NF-{kappa}B activation and COX-2 expression. These effects were observed in the absence of exogenous progesterone, suggesting a ligand-independent action of PR. Based on these findings, we propose that PR may inhibit NF-{kappa}B activation of COX-2 gene expression and uterine contractility via ligand-dependent and ligand-independent mechanisms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DURING APPROXIMATELY 95% of human pregnancy, the uterus is maintained in a state of almost complete quiescence by elevated circulating levels of progesterone. In women, circulating progesterone and myometrial levels of the progesterone receptor (PR) remain elevated throughout pregnancy and into labor. Thus, spontaneous labor is likely mediated by a concerted series of biochemical events that negatively impact the ability of the PR to regulate target genes that maintain myometrial quiescence.

Both term and preterm labor are associated with increased concentrations of cytokines (i.e. IL-1ß) in amniotic fluid (1), infiltration of the myometrium by neutrophils and macrophages (2, 3), and activation of nuclear factor-{kappa}B (NF-{kappa}B) in the myometrium (3, 4). Activated NF-{kappa}B, in turn, increases expression of genes promoting myometrial contractility, including the prostaglandin F2{alpha} receptor (5), the gap junction protein connexin 43 (6), the oxytocin receptor (7), and cyclooxygenase-2 (COX-2) (8). We previously observed that surfactant protein A (SP-A) secreted by the fetal mouse lung into amniotic fluid after gestation d 17, provides a key hormonal stimulus for the cascade of inflammatory signaling pathways within the maternal uterus that culminate in the enhanced myometrial contractility leading to parturition (3).

Uterine quiescence throughout pregnancy is maintained by progesterone acting through its nuclear receptor. In the majority of mammalian species, transition of the pregnant uterus from a state of near quiescence to an activated state of responsiveness to contractile stimuli is associated with a precipitous decline in circulating levels of progesterone (9). By contrast, in humans, circulating levels of progesterone remain elevated throughout gestation and into labor, as do levels of myometrial PR (10). In consideration of the high affinity of progesterone for its receptor, it is questionable as to whether the marked decline in circulating progesterone that occurs in other mammals (9) reaches levels that compromise its actions at receptors within the uterus and cervix. The finding that treatment of pregnant women (11) and guinea pigs with PR antagonists results in cervical ripening and increased sensitivity of the uterus to contractile hormones suggests that progesterone acting through PR plays an important role in maintaining uterine quiescence. Consequently, we postulate that spontaneous labor is initiated by a complex and concerted series of biochemical events that antagonize the ability of the PR to regulate target genes in the uterus that maintain myometrial quiescence. However, to date, the PR-responsive genes that block uterine contractility remain to be identified, as are the mechanisms that compromise PR function at term.

In recent studies, some of the mechanisms involved in the withdrawal in PR function associated with labor have been elucidated. We observed that parturition in humans and mice was associated with a pronounced decline in uterine levels of PR coactivators containing histone acetylase activity and that treatment of pregnant mice with a histone deacetylase inhibitor delayed the onset of labor (12). Moreover, a putative corepressor that promotes PR degradation and inhibits its transcriptional activity has been reported to increase in pregnant rat myometrium at term (13). The initiation of labor in mice also is accompanied by an increase in the local metabolism of progesterone in both the uterus (14) and cervix (15, 16). Furthermore, the onset of labor in humans and mice was found to be associated with enhanced expression of the inhibitory PR isoforms, PR-A (17) and PR-C (4).

Although strong evidence supports the existence of a stimulatory pathway leading to labor and a distinct pathway maintaining myometrial quiescence, it is possible that both are not mutually exclusive. Evidence exists to support the notion that progesterone may directly or indirectly impair the NF-{kappa}B-mediated inflammatory cascade leading to labor. First, it has been demonstrated that a mutual negative interaction exists between the PR and NF-{kappa}B p65 in both COS-1 and HeLa cells (18). Second, studies by Condon et al. (4) have shown that activation of NF-{kappa}B in mouse uterus and in human myometrial cells promotes enhanced expression of inhibitory PR isoforms. Finally, in human lower uterine segment (LUS) fibroblasts and in amnion epithelial cells, progesterone was found to repress NF-{kappa}B transcriptional activity (19).

The focus of this study was to determine whether progesterone/PR can antagonize NF-{kappa}B-mediated gene expression within the context of the human myometrium, and to define the molecular mechanisms involved. To accomplish this end, we used cyclooxygenase-2 (COX-2) as a candidate gene because it contains two well-characterized NF-{kappa}B response elements in its promoter (8, 20, 21) and is expressed in the uterus throughout pregnancy and labor in the majority of mammals (22, 23, 24, 25). We also examined cyclooxygenase-1 (COX-1) as a negative control because previous findings indicate the absence of functional NF-{kappa}B response elements in its promoter (26). Herein, we have elucidated both ligand-dependent and ligand-independent mechanisms whereby PR suppresses the IL-1ß-induced expression of COX-2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Progesterone Suppresses IL-1ß Induction of COX-2 Expression in Immortalized Human Myometrial Cells
COX-2 expression has been reported to increase in fundal myometrium of women in labor (22, 27). To examine the underlying mechanisms for COX-2 induction, immortalized human fundal myometrial cells (hTERT) that contain PR (4) were incubated in the absence or presence of IL-1ß (10 ng/ml) for up to 12 h ± progesterone (10–7 M). IL-1ß alone caused a marked up-regulation of COX-2 mRNA after 2 h of incubation, with maximal stimulation (~30 fold) after 6 h in culture (Fig. 1AGo). Treatment with progesterone suppressed IL-1ß induction at both 6 and 12 h of incubation. Progesterone alone had no effect on the basal levels of COX-2 at the time points examined. Although appreciable levels of COX-1 mRNA exist in these cells, neither IL-1ß nor progesterone had effects on COX-1 expression (data not shown).


Figure 1
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  		Fig. 1. Progesterone (P4) Impairs IL-1ß Induction of COX-2 Expression in a Time-Dependent Manner

hTERT myometrial cells were treated for 0.25–12 h in the absence or presence of 10 ng/ml IL-1ß with or without progesterone (10–7 M). RNA was isolated and the expression of COX-2 was analyzed by Q-RT-PCR. Data are the mean ± SEM of values from four independent experiments and are expressed as fold-induction over the control.

 
Progesterone Inhibits IL-1ß-Induced Recruitment of NF-{kappa}B p65 to Both the Proximal and Distal NF-{kappa}B Elements within the COX-2 Gene Promoter in Immortalized Human Myometrial Cells
In these studies, chromatin immunoprecipitation (ChIP) was used to examine the time-dependent effects of progesterone and IL-1ß on in vivo binding of NF-{kappa}B p65 to both the proximal (–223) and distal (–447) NF-{kappa}B elements of the COX-2 promoter. Human myometrial cells were cultured in the absence or presence of IL-1ß with or without progesterone for 2 or 6 h. After immunoprecipitation of chromatin complexes with antiserum against p65, quantitative real-time PCR was used to amplify an approximately 100-bp genomic region surrounding each of the two NF-{kappa}B response elements within the 5'-flanking region of the COX-2 gene (Fig. 2Go). The primers were demonstrated to amplify their target sequences with equal efficiency over a wide range of chromatin concentrations (data not shown). As can be seen in Fig. 2Go, A and B, IL-1ß stimulated recruitment of p65 to both NF-{kappa}B response elements, with greater amounts of p65 bound to the proximal NF-{kappa}B element. IL-1ß stimulation of p65 binding was maximal after 2 h of incubation. Notably, cotreatment with progesterone decreased the recruitment of p65 to both the proximal and distal NF-{kappa}B response elements at both time points.


Figure 2
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  		Fig. 2. Progesterone (P4) Impairs IL-1ß-Induced Recruitment of NF-{kappa}B p65 to Both the Proximal and Distal NF-{kappa}B Response Elements of the COX-2 Gene Promoter

hTERT myometrial cells were cultured for 2 or 6 h in serum-free medium with or without IL-1ß (10 ng/ml) and progesterone, added alone and in combination. The cells were then treated with 1% formaldehyde, followed by lysis and sonication to shear and solubilize the cross-linked chromatin, which was immunoprecipitated using an antibody specific for NF-{kappa}B p65. DNA was isolated, and the relative abundance of a 100-bp region surrounding both the distal (panel A; –447) and proximal (panel B; –223) NF-{kappa}B response elements was quantified by real-time PCR using primers indicated in Table 1Go. Shown is a representative of three independent experiments with comparable results. The data are expressed as arbitrary units. The bars represent the mean ± SEM of values from three sets of culture dishes.

 
Progesterone Up-Regulates Expression of the NF-{kappa}B Inhibitor, I{kappa}B{alpha}, in Immortalized Human Myometrial Cells
To begin to decipher the mechanisms for the progesterone-associated decreases in NF-{kappa}B p65 binding to the NF-{kappa}B response elements of the COX-2 promoter in human myometrium, we analyzed expression levels of I{kappa}B{alpha}, a protein that blocks NF-{kappa}B transactivation, in the hTERT myometrial cells. As can be seen in Fig. 3AGo, I{kappa}B{alpha} mRNA levels were rapidly induced by progesterone. This was evident after as early as 15 min of incubation, with maximal effects evident by 2 h (Fig. 3AGo). I{kappa}B{alpha} protein levels also were analyzed in the myometrial cells by immunoblotting. After 6 h of incubation, the time at which progesterone exerted a maximal inhibitory effect on COX-2 expression, immunoblotting for I{kappa}B{alpha} revealed that progesterone caused a pronounced increase in I{kappa}B{alpha} protein levels (Fig. 3BGo). As expected, IL-1ß treatment markedly reduced I{kappa}B{alpha} protein levels, presumably by enhancing I{kappa}B{alpha} degradation via the proteasome pathway (28). Interestingly, in cells coincubated with progesterone, degradation of I{kappa}B{alpha} in response to IL-1ß treatment was prevented (Fig. 3BGo).


Figure 3
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  		Fig. 3. Progesterone (P4) Rapidly Induces Expression of I{kappa}B{alpha} in hTERT Myometrial Cells

A, hTERT myometrial cells were treated for 0.25, 2, 6, or 12 h in the absence or presence of 100 nM progesterone. RNA was isolated and the expression of I{kappa}B{alpha} mRNA was analyzed by Q-RT-PCR. Data are the mean ± SEM of values from four independent experiments and are expressed as arbitrary units. B, hTERT myometrial cells were cultured in the absence or presence of IL-1ß (10 ng/ml) with or without progesterone for 6 h; cytoplasmic extracts were analyzed for I{kappa}B{alpha} protein levels by immunoblotting. The experiment was repeated three times with similar results. Veh, Vehicle.

 
Ablation of the PR Subtypes A and B by PR Small Interfering RNA (siRNA) Markedly Induces Basal Expression of COX-2 in T47D Breast Cancer Cells
To examine whether progesterone inhibition of COX-2 mRNA is PR subtype specific, T47D cells, which express all three PR isoforms, were transfected with a siRNA specific for full-length PR (PR-B), or with siRNAs for both PR-B and the truncated PR-A, or with an PR-B siRNA mismatch, as control (Fig. 4AGo). Cells incubated with the transfection reagent in the absence of siRNA were also studied as control (Lipid). The T47D cells were chosen for these experiments in lieu of the hTERT myometrial cells because, unlike the hTERT cells, T47D cells can be efficiently transfected with the PR siRNAs to achieve complete knockdown of PR-A and PR-B proteins. After 4 d of culture, the cells were incubated for 6 h in the absence or presence of IL-1ß (10 ng/ml), progesterone (100 nM), or the two agents in combination, and COX-2 and COX-1 mRNA levels were analyzed by quantitative RT-PCR (Q-RT-PCR) (Fig. 4Go, B and C). As observed in the studies shown in Fig. 1Go, in control cells transfected with PR-B siRNA mismatch or with transfection reagent alone, IL-1ß stimulated COX-2 expression, whereas progesterone diminished this induction (Fig. 4BGo). Reduction of PR-B protein levels using siRNA specific for PR-B mRNA did not alter basal COX-2 mRNA levels or the ability of progesterone to impair IL-1ß-induced increases in COX-2 expression, as compared with cells incubated with transfection reagent alone or transfected with an siRNA mismatch. By contrast, ablation of PR-A plus PR-B using siRNA directed against both PR subtypes greatly enhanced both basal and IL-1ß-stimulated COX-2 mRNA (Fig. 4BGo). This was associated with a pronounced increase in nuclear NF-{kappa}B p65 protein levels (Fig. 4AGo). Furthermore, in the T47D cells lacking PR-A and PR-B, progesterone was unable to diminish COX-2 expression (Fig. 4BGo). Moreover, none of the PR siRNA treatments had an effect on basal expression of I{kappa}B{alpha} protein (Fig. 4AGo). Additionally, transfection of T47D cells with PR-A + PR-B siRNAs had no effect on the basal expression of the COX-1 gene (Fig. 4CGo), which lacks functional NF-{kappa}B response elements in its promoter.


Figure 4
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  		Fig. 4. Knockdown of PR-A and PR-B Markedly Enhances Expression of COX-2 mRNA in T47D Cells

T47D cells were transfected with siRNA targeted against PR-B alone or with siRNAs against both PR-B and PR-A for 5 d. Cells were cultured for 6 h in the absence or presence of 10 ng/ml IL-1ß with or without progesterone (10–7 M). Cell lysate proteins were analyzed for levels of PR, NF-{kappa}B p65, I{kappa}B{alpha}, and ß-actin by immunoblotting (panel A). RNA was isolated and the expression of COX-2 (panel B), and COX-1 (panel C) mRNA transcripts were analyzed by Q-RT-PCR. Data are the mean ± SEM of values from four independent experiments and are expressed as arbitrary units. MM, Mismatch; MisM, mismatch; P4, progesterone.

 
Coincubation with the Antiprogestin RU486 Prevents the Progesterone-Induced Suppression of COX-2 mRNA in hTERT Myometrial and in T47D Cells
Given that siRNA-induced knockdown of PR-A and PR-B in T47D cells enhanced basal and IL-1ß-stimulated COX-2 expression and prevented progesterone-induced suppression of COX-2, we wanted to further address whether the antiprogestin RU486 would yield similar results in the T47D and in hTERT myometrial cells. As can be seen in Fig. 5Go, RU486 had no effect on COX-2 mRNA levels in control, IL-1ß-, or in progesterone-treated hTERT myometrial or T47D cells. However, RU486 effectively blocked progesterone-mediated inhibition of COX-2 mRNA in both cell types (Fig. 5Go, A and B).


Figure 5
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  		Fig. 5. Coincubation with RU486 Blocks the Progesterone (P4)-Induced Suppression of COX-2 mRNA in Myometrial and T47D Cells

hTERT (panel A) or T47D cells (panel B) were incubated for 6 h in the absence or presence of RU486 (10–8 M for hTERT cells and 10–7 M for T47D cells) with and without IL-1ß (10 ng/ml), progesterone (10–7 M), or both. RNA was isolated and the expression of COX-2 was analyzed by RT-Q-PCR. The data are expressed as fold increase over vehicle-treated control cells. The bars represent the mean ± SEM of values from three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, we made the notable observation that progesterone acting through PR plays a major antiinflammatory role in human myometrial cells through antagonism of NF-{kappa}B activation and the subsequent induction of COX-2, the crucial and rate-determining enzyme in prostaglandin biosynthesis. We suggest that this mechanism underlies the action of progesterone/PR to maintain myometrial quiescence throughout pregnancy because of the critical role of prostaglandins in uterine contractility during labor (29).

In recent studies by our laboratory (27) and others (22), COX-2 mRNA was found to be up-regulated in fundal myometrium of women during labor. To date, the majority of studies to investigate changes in expression of cyclooxygenases in the myometrium of women in labor vs. not in labor at term have demonstrated little, if any, increase in myometrial COX-2 associated with labor (30, 31, 32, 33); however these investigations were primarily carried out using LUS, rather than fundal myometrium. The finding of a fundus-specific induction of COX-2 mRNA (22) is significant, because during labor contractility occurs within the fundus, while the LUS myometrium relaxes to allow expulsion of the fetus (34). On the other hand, previous microarray studies have demonstrated that there are no alterations in COX-1 expression with labor (30). This is not surprising, because the COX-1 gene is constitutively regulated and lacks functional NF-{kappa}B response elements (26).

The observed increases in COX-2 expression in fundal myometrium of women in labor are particularly intriguing in light of recent findings from our laboratory that both NF-{kappa}B activation and the induction of inhibitory PR-C isoform expression occur during labor in human fundal myometrium and are not apparent in the LUS (4). Increased expression of COX-2 with the onset of labor also has been observed in a number of other species (24, 25). In mice, uterine COX-2 was found to be markedly induced at 19 d post coitum, concomitant with increased expression of connexin-43 and the oxytocin receptor (35). We also observed that COX-2 mRNA levels were dramatically increased in the pregnant mouse uterus at 19 d post coitum in association with labor (27); this was accompanied by increases of the ratios of the inhibitory PR isoforms, PR-A and PR-C, relative to the PR-B isoform (4).

In the present study, using immortalized hTERT fundal myometrial (36) and T47D breast cancer cells, we observed that IL-1ß increased COX-2 mRNA within 6 h of treatment and that progesterone caused a dose-dependent impairment of this induction. A similar phenomenon was observed in other studies using human lung type II cells (37), amnion epithelial cells, and lower uterine segment fibroblasts (19). To begin to define the molecular mechanisms underlying the progesterone-induced impairment of COX-2 expression, we employed ChIP to examine whether progesterone alters the in vivo binding of NF-{kappa}B p65 to the COX-2 promoter. In agreement with recent studies by Soloff et al. (8), we observed that treatment of hTERT myometrial cells with IL-1ß enhanced in vivo recruitment of NF-{kappa}B p65 to both the proximal (–233) and distal NF-{kappa}B (–447) response elements of the COX-2 promoter. Using real-time PCR primers that specifically targeted each NF-{kappa}B response element with the same efficiency, we extended their initial observations by demonstrating that there is greater in vivo binding of p65 to the proximal promoter as compared with the distal promoter after IL-1ß treatment. Importantly, we found that progesterone treatment impaired p65 in vivo binding to both proximal and distal promoters. These findings indicate that progesterone acting through PR antagonizes cytokine-induced COX-2 gene transcription. The progesterone-mediated decrease in p65 binding to the COX-2 promoter might be caused, in part, by a direct physical interaction of PR with p65 as was previously observed in vitro (18), resulting in a repression of NF-{kappa}B DNA-binding and transcriptional activity.

Notably, we have obtained compelling evidence for a ligand-independent action of PR to block NF-{kappa}B activation and expression of COX-2 in T47D cells, which were used in lieu of the myometrial cells because of the ease of transfectability of this breast cancer cell line. We reasoned that T47D cells were an appropriate alternative to myometrial cells because we observed that progesterone equivalently antagonized IL-1ß induction of COX-2 expression (Figs. 1Go, 4Go, and 5Go) and that the antiprogestin RU486 could reverse this inhibition (Fig. 5Go) in both cell lines. Furthermore, we have previously observed that IL-1ß up-regulated levels of nuclear NF-{kappa}B and PR proteins in both the hTERT myometrial and T47D cells (4). Such findings suggest that, in both cell lines, common mechanisms exist for the antiinflammatory actions of progesterone and for the up-regulation of PR expression by inflammatory cytokines. In these studies, siRNAs specific for PR-B and for PR-A plus PR-B were used to determine which PR subtype mediates inhibition of COX-2 in T47D cells. Whereas siRNA directed against PR-B alone had no effect on NF-{kappa}B activation or on COX-2 mRNA levels, complete knockdown of PR-A plus PR-B enhanced NF-{kappa}B p65 nuclear translocation and caused a more than 30-fold increase in COX-2 mRNA levels. These effects were observed in the absence of exogenous progesterone treatment and prevented progesterone-mediated inhibition of COX-2 expression. Although these findings suggest an important role of PR-A as an antagonist of NF-{kappa}B up-regulation of COX-2 expression, they do not discount the importance of PR-B, because the PR-B-specific siRNA did not completely eliminate PR-B protein within the cultured cells. Although the siRNA experiments could not effectively be carried out in hTERT myometrial cells, we postulate that PR may act in a similar ligand-independent manner in the pregnant uterus. Similarly, in HeLa and COS-1 cells, both overexpressed PR-B and PR-A were found to impair p65-mediated transactivation in a ligand-independent manner (18).

These findings indicate that the PR exerts a dominant ligand-independent inhibitory action on COX-2 expression, which can be overcome, in part, by activation of the NF-{kappa}B pathway by IL-1ß. In addition, when PR-A and PR-B were ablated in the siRNA-treated T47D cells, progesterone was no longer able to repress IL-1ß-induced increases in COX-2, indicating that progesterone must act through the PR to mediate its inhibitory effects. Moreover, the finding that treatment with RU486 failed to enhance IL-1ß-induced COX-2 expression in the absence of added progesterone in hTERT and T47D cells further supports the existence of distinct ligand-dependent and -independent actions of PR to inhibit NF-{kappa}B-mediated induction of COX-2 expression. Whereas the significance of a ligand-independent inhibitory role of PR on NF-{kappa}B activation in the pregnant uterus at term may seem unlikely in light of the high concentrations of circulating progesterone, our findings clearly indicate that PR exerts an equivalent tonic inhibitory effect on NF-{kappa}B activation and COX-2 expression in the absence or presence of ligand. The effect of PR to inhibit NF-{kappa}B activation likely occurs by direct interaction with p65 (18). This ligand-independent mechanism may be of great importance in endometrial and breast tissues of postmenopausal women, where PR may serve a protective role in the presence of negligible circulating levels of progesterone. On the other hand, progesterone clearly is required for PR induction of I{kappa}B{alpha} expression.

Collectively, these observations suggest that the regulation of PR and NF-{kappa}B-mediated genes are functionally interrelated. On one hand, PR may promote uterine quiescence throughout pregnancy both through activation of genes that promote myometrial relaxation, and by preventing NF-{kappa}B activation of contractile genes, such as COX-2. On the other hand, at term, increased cytokines and NF-{kappa}B activation cause an up-regulation of genes that promote uterine contractility and a corresponding decline in PR function. In recent studies, we found that activation of NF-{kappa}B in pregnant mouse uterus by intraamniotic injection of SP-A, and in T47D breast cancer cells by IL-1ß treatment, caused an up-regulation of the inhibitory PR-C isoform (4). Additionally, the decreased expression of PR coactivators in human and mouse uterus at term (12) may be induced by cytokine-mediated activation of inflammatory response pathways (38). This decline in PR function, in turn, may further enhance NF-{kappa}B activation of contractile genes, such as COX-2.

Furthermore, in the present study, we also obtained convincing evidence that the antiinflammatory effect of progesterone within the myometrium is mediated, in part, by increased expression of I{kappa}B{alpha}, a crucial inhibitor of NF-{kappa}B transactivation. Progesterone caused a rapid induction of I{kappa}B{alpha} mRNA and protein expression in the immortalized myometrial cells, which preceded its effect to inhibit IL-1ß-induced COX-2 expression. Moreover, coincubation with progesterone prevented the IL-1ß-mediated decline in I{kappa}B{alpha} protein levels, suggesting an effect of progesterone/PR to block I{kappa}B{alpha} degradation via the proteasome pathway (28). An action of progesterone to inhibit NF-{kappa}B activation by induction of I{kappa}B{alpha} was previously observed in macrophage cell lines (39) and in T47D cells (40). The identification of I{kappa}B{alpha} as a progesterone-induced gene in the myometrial cells is of great interest considering that, to date, very few myometrial PR target genes have been identified. Furthermore, it is likely that I{kappa}B{alpha} serves as a crucial PR target gene that mediates myometrial quiescence during pregnancy because of its important role as an inhibitor of NF-{kappa}B activation and, thus, of genes that regulate myometrial contractility. The finding that siRNA-mediated ablation of both PR-A and PR-B had no effect on I{kappa}B{alpha} expression suggests that PR action to induce I{kappa}B{alpha} expression is ligand dependent and likely occurs via progesterone/PR induction of I{kappa}B{alpha} promoter activity.

The molecular mechanisms that mediate progesterone induction of I{kappa}B{alpha} gene expression have not been defined. Glucocorticoids acting through the glucocorticoid receptor (GR) are known to induce I{kappa}B{alpha} expression in a cell type- and promoter-specific manner (41, 42). It is conceivable that glucocorticoids and progestins may induce I{kappa}B{alpha} expression through similar mechanisms, because the GR and PR are structurally homologous and bind to a common response element in DNA [glucocorticoid response element/progesterone response element (GRE/PRE)]. Although no consensus GRE/PRE(s) have been identified in the 5'-flanking region of the I{kappa}B{alpha} gene (43), a reporter construct containing 623 bp of I{kappa}B{alpha} 5'-flanking sequence was sufficient for glucocorticoid induction of I{kappa}B{alpha} promoter activity in transfected cells (44). This region included a GRE half-site that was suggested to mediate glucocorticoid/GR effects to enhance binding of other activating transcription factors, including NF-{kappa}B, Ets-1, and Sp1 (43). Studies are planned to further define PR isoforms and mechanisms for progesterone induction of I{kappa}B{alpha} expression.

In conclusion, we have identified a novel role for PR as a potent antiinflammatory factor in the myometrium. Based on our findings, we propose that during most of pregnancy when PR is functionally active, elevated progesterone levels mediate PR induction of I{kappa}B{alpha}, which suppresses NF-{kappa}B activation of contractile genes, such as COX-2. The functionally active PR also directly inhibits COX-2 gene expression by blocking the binding of NF-{kappa}B to the COX-2 promoter. However, at term, the activation of macrophage migration and of inflammatory response pathways in the pregnant uterus by secretion of SP-A from fetal lung into amniotic fluid (3) causes increased NF-{kappa}B activation of contractile genes, including COX-2. The activation of inflammatory response pathways also may cause a decline in PR coactivators (12) and induction of inhibitory PR isoforms (4), resulting in a decline in PR function. This, in turn, may result in decreased capacity of progesterone/PR to activate I{kappa}B{alpha} expression and of PR to inhibit NF-{kappa}B activation by direct protein-protein interaction, resulting in an enhanced induction of COX-2, which culminates in the initiation of labor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Lines
Human breast cancer T47D cells were maintained in RPMI medium with 10% fetal bovine serum and 0.1 mM insulin. Human myometrial hTERT cells (36) were maintained in DMEM-F12 (Life Technologies, Gaithersburg, MD) with 10% fetal bovine serum. For experiments, cells were switched to serum-free medium and treated either with IL-1ß (10 ng/ml; Sigma Chemical Co., St. Louis, MO), progesterone (1–1000 nM; Sigma), or both, for 0–24 h. In some experiments, the cells also were incubated with RU486 (100 nM; Roussel Uclaf, Romainville, France). Cell lysates and nuclear extracts were prepared and analyzed by immunoblotting as described below.

Q-RT-PCR
Total RNA from hTERT myometrial and T47D breast cancer cells was extracted by the one-step method of Chomczynski and Sacchi (45) (TRIzol, Invitrogen, Carlsbad, CA). RNA was treated with deoxyribonuclease to remove any contaminating DNA, and 4 µg were reversed transcribed using random primers and Superscript II RNase H-reverse transcriptase (Invitrogen). Primer sets directed against human COX-1, COX-2, and I{kappa}B{alpha}, along with the constitutively expressed cyclophilin, were generated utilizing Primer Express software (PE Applied Biosystems, Boston, MA) based on published sequences (Table 1Go).


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  		Table 1. Primers Used in the Q-RT-PCR Analysis of Human Cell Lines

 
The relative abundance of each transcript was determined by real-time quantitative PCR using a modification of previously published methods (4). For the quantitative analysis of mRNA expression, the ABI Prism 7700 Detection System (Applied Biosystems, Foster City, CA) was employed using the DNA binding dye SYBER Green (PE Applied Biosystems) for the detection of PCR products. The cycling conditions were 50 C for 2 min, 95 C for 10 min, followed by 40 cycles of 95 C for 15 sec, and 60 C for 1 min. The cycle threshold was set at a level at which the exponential increase in PCR amplification was approximately parallel between all samples. All primer sets produced amplicons of the expected size and sequence. We calculated the relative fold changes using the comparative cycle times (Ct) method with cyclophilin as the reference guide. Over a wide range of known cDNA concentrations, all primer sets were demonstrated to have good linear correlation (slope = –3.4) and equal priming efficiency for the different dilutions compared with their Ct values (data not shown). Given that all primer sets had equal priming efficiency, the {Delta}Ct values (primer – internal control) for each primer set were calibrated to the experimental samples with the lowest transcript abundance (highest Ct value), and the relative abundance of each primer set compared with calibrator was determined by the formula, 2{Delta}{Delta}Ct, in which {Delta}{Delta}Ct is the calibrated Ct value.

ChIP
Immortalized human myometrial cells were cultured for up to 6 h in serum-free medium, in the absence or presence of IL-1ß (10 ng/ml), progesterone (100 nM), or the two hormones in combination. ChIP was performed using a modification of previously published methods (4, 46). Briefly, myometrial cells (3 – 1 x 107 cells per treatment) were washed once with PBS and incubated with 1% formaldehyde (in control medium) for 10 min at room temperature to cross-link proteins and DNA. Cross-linking was terminated by the addition of glycine (0.125 M, final concentration). The cells were washed twice with cold PBS and placed in 500 µl of lysis buffer [50 mM Tris (pH 8.1), 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail (Roche, Indianapolis, IN), and 5 mM EDTA]. The lysates were sonicated on ice to produce sheared, soluble chromatin. The soluble chromatin was precleared with Protein A/G Plus agarose beads (60 µl) at 4 C for 1 h. The samples were microfuged at 14,000 rpm to pellet the beads, and the supernatant containing the sheared chromatin was placed in new tubes. The precleared chromatin was aliquoted into 300 µl amounts and incubated with antibodies for p65 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 C overnight. Two aliquots were reserved as controls: one was incubated without antibody and the other with nonimmune IgG. Protein A/G Plus agarose beads (60 µl) were added to each tube, the mixtures were incubated for 2 h at 4 C, and the immune complexes were collected by centrifugation. The beads containing the immunoprecipitated complexes were washed sequentially for 5–10 min in wash buffer I (20 mM Tris-HCl, pH 8.1; 2 mM EDTA; 0.1% SDS; 1% Triton X-100; 150 mM NaCl), wash buffer II (same as wash buffer I, except containing 500 mM NaCl), wash buffer III (10 mM Tris-HCl, pH 8.1; 1 mM EDTA; 1% Nonidet P-40; 1% deoxycholate; 0.25 M LiCl), and in 2x Tris-EDTA buffer. The beads were eluted with 250 µl elution buffer [1% SDS, 0.1 mM NaHCO3 + 20 µg salmon sperm DNA (Sigma) at room temperature]. This was repeated once and eluates were combined. Cross-linking of the immunoprecipitated chromatin complexes and input controls (10% of the total soluble chromatin) was reversed by heating the samples at 65 C for 4 h. Proteinase K (15 µg, Invitrogen) was added to each sample in buffer (50 mM Tris-HCl, pH 8.5; 1% SDS; 10 mM EDTA) and incubated for 1 h at 45 C. The DNA was purified by phenol-chloroform extraction and precipitated in EtOH overnight at –20 C. Samples and input controls were diluted in 10–100 µl Tris-EDTA buffer just before PCR. Real-time PCR was employed using forward (5'-TAAAACATGTCAGCCTTTCTTAACCTT-3') and reverse (5'-CGGCCCTGAGGTCCG-3') primers that amplify an approximately 100-bp region surrounding the proximal NF-{kappa}B response element, and forward (5'-GGAGGAGAGGGAGGGATCAG-3') and reverse (5'-TGCCCCAATTTGGGAGC-3') primers surrounding the distal NF-{kappa}B response element of the COX-2 promoter. Using serial dilutions of human chromosomal DNA, these primers were demonstrated to have equal efficiency in priming their target sequences.

Lipid-Mediated Transfection of siRNA Duplexes
RNA oligonucleotides directed against PR-B, PR-A plus PR-B, or mismatch PR-B (Table 2Go) were purchased from The Center for Biomedical Inventions at The University of Texas Southwestern Medical Center at Dallas, and transfection was performed as described previously (47). Briefly, T47D cells were plated in medium lacking antibiotics at approximately 80,000 cells per well in six-well plates 2 d before transfection so that they would be 30–50% confluent at the time of transfection. On the day of transfection (d 0), siRNA duplexes (200 nM) and Oligofectamine (8 µl per well; Invitrogen) were diluted in Optimem (Invitrogen) according to the manufacturer’s instructions. The medium was changed 24 h later (d 1). On d 5, cells were placed in serum-free medium and treated with either IL-1ß (10 ng/ml) (Sigma), progesterone (100 nM) (Sigma), or both, for 6 h. RNA and/or cell lysates were prepared as described previously and analyzed by Q-RT-PCR and immunoblotting, respectively (4).


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  		Table 2. siRNAs Used

 
Preparation of Cell Lysates and Nuclear Extracts and Immunoblot Analysis
hTERT myometrial and T47D cell cytoplasmic and nuclear extracts were prepared as described previously (4). Briefly, human myometrial tissue and cultured cell cytoplasmic extracts were prepared by homogenizing the tissue or cells in ice-cold buffer containing 10 mM HEPES (pH 7.5), 10 mM MgCl2, 5 mM KCl, and 0.1% Triton X-100. The homogenate was centrifuged at 5000 rpm for 10 min at 4 C, and the supernatant was retained as the cytoplasmic fraction; the remaining pellet was further processed for nuclear extraction. Nuclear extracts were prepared by resuspending the pellet in ice-cold buffer containing 25% glycerol, 20 mM HEPES (pH 7.9), 500 µM NaCl, 1.5 mM MgCl2, and 0.2 mM EDTA (pH 8.0). The resuspended pellet was incubated on ice for 30 min, vortexed every 5 min, and centrifuged at 10,000 rpm for 10 min at 4 C, and the supernatant was retained as the nuclear fraction (48). Equal concentrations of nuclear and cytoplasmic proteins, normalized by colorimetric BCA Protein Assay (Pierce Corp., Madison, WI), were fractionated in gradient polyacrylamide gels (Invitrogen) and transferred onto Hybond-P (Amersham Pharmacia Biotech, Arlington Heights, IL). Blots were probed using rabbit antibodies for PR (Cell Signaling Technology, Beverly, MA), which recognizes PR-A and PR-B isoforms, I{kappa}B{alpha} (1:1000) (sc-203, Santa Cruz Biotechnology), and NF-{kappa}B p65 (C-20): sc-372 (Santa Cruz Biotechnology), diluted in 5% milk-1x Tris-buffered saline-Tween 20 buffer and with horseradish peroxidase-conjugated goat antirabbit IgG (1:10,000) diluted in 5% milk-1x Tris-buffered saline-Tween 20 buffer (Amersham Pharmacia Biotech) as the secondary antibody. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech).


   FOOTNOTES
 
This work was supported by: National Institutes of Health Grants 5 P01 HD011149 (to C.R.M.); 5 R01 GM 60642 and 5 R01 GM 73042 (to D.R.C.); Research Grants 21-FY04–174 from the March of Dimes Birth Defects Foundation (to C.R.M.); and I-1244 from the Robert A. Welch Foundation (to D.R.C.). D.B.H. is a recipient of a postdoctoral fellowship (PDF0600877) from the Susan G. Komen Breast Cancer Foundation.

None of the authors (D.B.H., B.J., D.C., and C.R.M.) has anything to declare regarding potential conflicts of interest.

First Published Online June 13, 2006

Abbreviations: ChIP, Chromatin immunoprecipitation; COX-2, cyclooxygenase-2; Ct, cycle time; GR, glucocorticoid receptor; GRE, glucocorticoid response element; hTERT, human fundal myometrial cells; I{kappa}B{alpha}, inhibitor of {kappa}B{alpha}; LUS, lower uterine segment; NF-{kappa}B, nuclear factor {kappa}B; PR, progesterone receptor; PR-A, PR-B, PR-C, PR isoforms; PRE, progesterone response element; Q-RT-PCR, quantitative RT-PCR; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; SP-A, surfactant protein A.

Received for publication March 7, 2006. Accepted for publication June 7, 2006.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   PR
Ligands:   Progesterone  |  RU486



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