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Molecular Endocrinology 20 (4): 764-775
Copyright © 2006 by The Endocrine Society

Up-Regulation of the Progesterone Receptor (PR)-C Isoform in Laboring Myometrium by Activation of Nuclear Factor-{kappa}B May Contribute to the Onset of Labor through Inhibition of PR Function

Jennifer C. Condon, Daniel B. Hardy, Kelly Kovaric and Carole R. Mendelson

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

Address all correspondence and requests for reprints to: Carole R. Mendelson, Ph.D., Department of Biochemistry, University of Texas Southwestern Medical Center, 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
 
Progesterone acting via the progesterone receptor (PR) plays a critical role in maintaining uterine quiescence during pregnancy. In the present study, we tested the hypothesis that the transactivating capability of the PR is down-regulated in the myometrium at term by a change in uterine PR isoform ratio resulting from local activation of the nuclear factor (NF)-{kappa}B pathway. Overexpression of the truncated PR-C isoform in human myometrial cells inhibited PR-B transactivation. Expression of PR isoforms, PR-A, PR-B, and PR-C, was characterized by immunoblotting and quantitative PCR (Q-PCR) in fundal and lower uterine segment myometrium from pregnant women in labor and not in labor and in the pregnant mouse uterus during late gestation. We observed a marked increase in levels of PR-C and transcriptionally active PR-B specifically in fundal myometrium of women in labor. In pregnant mouse uterus, levels of PR-B and PR-C also increased between 15 days post coitum and term, whereas expression of PR-A was dramatically up-regulated at 19 days post coitum. In studies of uterine tissues of mice injected intraamniotically with surfactant protein A and of human myometrial and T47D breast cancer cells in culture, up-regulation of PR isoform expression was observed in response to activation of the NF-{kappa}B pathway. Chromatin immunoprecipitation analysis revealed IL-1ß induced binding of NF-{kappa}B to the PR promoter. Collectively, these findings suggest that up-regulation of inhibitory PR isoform expression by NF-{kappa}B activation in both laboring human fundus and pregnant mouse uterus near term may inhibit PR transactivation and thereby lead to a loss of uterine quiescence and the onset of labor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PROGESTERONE PLAYS an important role in the maintenance of uterine quiescence throughout most of pregnancy by suppressing expression of genes that mediate increased myometrial contractility, such as the oxytocin receptor (1) and the gap junction protein connexin 43 (2). The decline in circulating levels of progesterone that accompanies the onset of labor in most mammalian species suggests that progesterone withdrawal is critical for the increase in uterine contractility leading to labor (3). However, in humans, circulating progesterone levels remain elevated throughout the third trimester of pregnancy and into the onset of labor (4). The finding that administration of the PR antagonist RU486, to pregnant women can increase uterine contractility and lead to the onset of labor signifies the importance of PR transactivation in the inhibition of uterine contractility and suggests that parturition is caused by molecular events that impair PR function (5).

In previous studies, we observed that parturition in humans and mice is associated with a pronounced decline in uterine levels of coactivators containing histone acetylase activity that are known to enhance PR transcriptional activity and that treatment of pregnant mice with a histone deacetylase inhibitor delayed the onset of labor (6). We also observed that surfactant protein A (SP-A) secreted by the fetal mouse lung into amniotic fluid after gestation d 17, activates fetal amniotic fluid macrophages, causing them to migrate to the uterine wall where they activate nuclear factor (NF)-{kappa}B, resulting in increased uterine contractility (7). We postulated that SP-A-induced activation of NF-{kappa}B increases uterine contractility by two mechanisms. On one hand, activated NF-{kappa}B increases expression of target genes that cause increased myometrial contractility, such as cyclooxygenase-2 (COX-2) (8, 9) (Hardy D. B., and C. R. and Mendelson, unpublished observations). On the other hand, NF-{kappa}B p65 may also antagonize PR activation of target genes that modulate uterine contractility by interacting with the PR and reducing its DNA binding and transcriptional activity (10). In this study, we propose that NF-{kappa}B activation may mediate changes in uterine PR isoform expression.

The biological actions of progesterone are mediated through the PR which is expressed as three isoforms, PR-A, PR-B, and PR-C. PR mRNAs are generated from a single gene by differential promoter use (11, 12). PR-B has been shown to function as a strong transactivator of progesterone-regulated genes; when PR-A and PR-B are coexpressed, the A-isoform can repress the action of PR-B (13, 14). The inhibitory action of PR-A protein was suggested to be due to its inability to efficiently recruit coactivators and its increased interaction with corepressors, as compared with PR-B (13). However, studies of gene targeted mice where PR-A or PR-B were independently ablated suggest that both isoforms function in a tissue-specific manner as distinct transcriptional activators (15, 16, 17). The third PR isoform, PR-C, an N-terminally truncated form of PR lacking the DNA binding domain with a molecular mass of about 60 kDa, has been reported to be restricted primarily to the cytosolic fraction (18). Given that PR-C does not have the capacity to bind DNA but is able to bind progesterone (19), PR-C may inhibit PR function by sequestering available progesterone away from the PR-B-isoform. PR-C has also been found to bind to the PR-B isoform, thereby reducing the capacity of PR-B to bind to PR response elements (20). As a result of isoform-specific functional differences, tissue responses to progesterone may be profoundly affected by changes in PR-A:PR-B:PR-C expression ratios.

The expression ratio of PR isoforms in the myometrium of women in labor as compared with those not in labor has previously been investigated (21, 22, 23). However, all of these studies failed to analyze the expression levels of PR-C, which we found in the present study to be markedly up-regulated in the pregnant uterus during labor. Moreover, all studies to date have been performed on myometrial biopsies isolated only from the lower uterine segment (LUS) (21, 22, 23), which relaxes during labor to allow expulsion of the fetus. Such findings are, therefore, unlikely to reflect molecular changes that mediate increased uterine contractility, which is manifest in the uterine fundus (24). Studies also have been performed using immunohistochemical analysis (21), but these studies fail to address changes in PR-isoform expression.

Herein we provide evidence that PR-C isoform expression increases dramatically both in pregnant mouse uterus and human fundal myometrium at term as a result of increased NF-{kappa}B activation and binding to the PR gene promoter. We propose that such alterations in the relative amount of PR-C to PR-B expressed in the pregnant uterus at term, blocks the capacity of PR-B to maintain uterine quiescence, resulting in increased uterine contractility. Moreover, our findings suggest that a withdrawal of PR function may occur locally in the pregnant uterus at term independent of circulating levels of progesterone, in both mice and humans.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transient Transfection of PR-C into Human Telomerase Reverse Transcriptase (hTERT) Cells Reduces Endogenous Levels of PR Transactivation
To confirm that PR-C functions as an inhibitory PR isoform hTERT-immortalized human myometrial (25) cells, which we have found in this study [by using an improved nuclear protein extraction protocol (26) to solely express the PR-B isoform, as discussed later (see Fig. 6Go)], were transiently cotransfected with a luciferase reporter vector under the control of the PR-responsive mouse mammary tumor virus (MMTV) long-terminal repeat (MMTV-Luc) (14), and increasing concentrations of a human PR (hPR)-C-containing expression vector (0.5, 1, and 1.5 µg). After overnight recovery, the cells were treated with vehicle (cnt), IL-1ß (10 ng/ml), or the PR antagonist RU486 (100 nM) for 12 h, lysed and assayed for reporter activity using a dual luciferase reporter assay system (Promega, Madison, WI). Transfection of the hTERT cell line with the MMTV-Luc reporter construct resulted in relatively high levels of reporter activity (Fig. 1Go). However, cotransfection of increasing amounts of the hPR-C expression vector resulted in a 4-fold decrease in luciferase activity as compared with the MMTV-Luc reporter construct alone. These results indicate that hTERT cells, which express PR-B alone, are capable of endogenous PR transactivation that can be compromised by transiently transfected hPR-C. Cytokine stimulation of hTERT cells by IL-1ß treatment further induced endogenous PR-B transcriptional activity. This was associated with IL-1ß induction of endogenous levels of PR-B as discussed later (see Fig. 6Go) and with a reduction in the inhibitory capacity of hPR-C (Fig. 1Go). However, transfected hPR-C maintained its ability to inhibit PR-B transactivation. In the presence of RU486, PR-B transactivation was ablated, and cotransfection of hPR-C expression vector had no additional effect (Fig. 1Go). Progesterone (100 nM) treatment inhibited endogenous PR-B expression and transcriptional activity, so that the inhibitory effect of cotransfected PR-C was less pronounced (data not shown).


Figure 6
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  		Fig. 6. PR-A, PR-B, and PR-C Expression Is Increased in Response to Activation and Decreased in Response to Inhibition of the NF-{kappa}B Pathway in Cultured hTERT Myometrial Cells and T47D Breast Cancer Cells

Nuclear extracts were prepared from T47D and hTERT myometrial cells that had been incubated for 12 h in the absence or presence of IL-1ß at 1, 5, 10, or 20 ng/ml (increasing wedge), or SN50 (50 µg/ml). NF-{kappa}B activation was assessed by immunoblot analysis of nuclear levels of p65. This experiment was repeated three times with similar results.

 

Figure 1
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  		Fig. 1. Overexpression of PR-C Causes a Dose-Dependent Decrease in Endogenous PR-B Transcriptional Activity in hTERT Myometrial Cells

hTERT cells that express endogenous PR-B as the only PR isoform were transiently cotransfected with MMTV-Luc (14 ) containing four PR response elements and a hPR-C expression vector (CMV:PRC) in increasing concentrations (0.5, 1, and 1.5 µg). Levels of transfected DNA were kept constant by cotransfection of the empty expression vector (EV). After overnight recovery, cells were treated with IL-1ß (10 ng/ml), the PR inhibitor RU486 (100 nM), or vehicle alone (Cnt) for 12 h, lysed and assayed for reporter activity. Results are expressed as the mean ± SEM of data from three independent experiments, each performed in triplicate.

 
Expression of the PR-B and Inhibitory PR-C Isoforms Increase in Fundal Myometrial Tissues from Women in Labor
Previous studies to analyze changes in PR isoform expression in human myometrium focused on the LUS (23) rather than on fundus, where uterine contractility is initiated. Furthermore in those studies, the primers used for Q-PCR to purportedly quantify PR-A + PR-B also amplified PR-C. As mentioned above, in the present study, we used primers that amplified with equivalent efficiency PR-B alone, PR-A + PR-B and PR-A + PR-B + PR-C and quantified PR-A and PR-C isoforms by subtraction. Nuclear and cytoplasmic proteins and total RNA isolated from fundus and lower uterine segment myometrium of women in labor and not in labor (n = 3 for each group) were analyzed for expression levels of all three PR isoforms by immunoblotting and Q-PCR, respectively. As is shown in Fig. 2AGo, protein levels of the PR-B isoform were readily detectable in LUS and fundal nuclear extracts of all tissues analyzed. PR-B protein levels were specifically increased in the fundal myometrium of women in labor. By contrast, no apparent change during labor in the levels of PR-B protein was detectable in the LUS myometrium. PR-A and PR-C protein levels were undetectable in all myometrial nuclear extracts. On the other hand, PR-C protein levels were remarkably increased in cytoplasmic fractions of fundal myometrium of women in labor compared with those not in labor, whereas PR-C was undetectable in cytoplasmic fractions from the LUS (Fig. 2BGo). An unidentified immunoreactive band larger than PR-C was detected in lane 11 of Fig. 2BGo. Based on its size, it is unlikely to be a phosphorylated or sumoylated form of PR-C or to be PR-A and is therefore likely to be an artifact. Neither PR-A nor PR-B protein were detectable in cytoplasmic fractions of the LUS or fundus (data not shown). This is in contrast to T47D breast cancer cells, in which PR-A, PR-B, and PR-C proteins were readily detectable in isolated nuclear fractions (as shown later, see Fig. 4AGo).


Figure 2
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  		Fig. 2. PR-B and PR-C Isoform Expression Is Up-Regulated in the Fundal Myometrium of Women in Labor

A, Nuclear (nuc) and cytoplasmic (cyto) extracts (11 µg) isolated from LUS (1–6) and fundal (7–12) myometrium of three women not in labor (NIL) (1–3, 7–9) and three women in labor (IL) (4–6, 10–12) (n = 3 for each group) were analyzed for PR-A, PR-B and PR-C expression by immunoblotting (top panel). B, Cytoplasmic extracts (20 µg) isolated from LUS (1–6) and fundal (7–12) myometrium of three women not in labor (1–3, 7–9) and in labor (4–6, 10–12) (n = 3 for each group) were analyzed for PR-A, PR-B, and PR-C expression by immunoblotting (top panel).

 

Figure 4
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  		Fig. 4. The Inhibitory PR Isoforms PR-C and PR-A Are Up-Regulated in the Pregnant Mouse Uterus in Late Gestation

A, Nuclear and cytoplasmic extracts from pregnant mouse uterus at 15–19 dpc were analyzed for PR-A, PR-B, and PR-C expression by immunoblotting. PR-B levels increased between 15 and 19 dpc. The immunoblot shown is representative of findings obtained using uteri from three different gestational series of mice. B–D, RNA isolated from uteri of three series of pregnant mice at 15–19 dpc were analyzed for PR-B, PR-A + B and PR-A + B + C mRNA by Q-PCR using oligonucleotides that primed with equivalent efficiency. Levels of PR-B, PR-A, and PR-C mRNA were ascertained by subtraction. Data are the mean ± SEM of values from three gestational series of pregnant mice. Expression levels of all three PR isoforms increased toward term.

 
Q-PCR mRNA analysis of myometrial tissues from the same subjects demonstrated that expression of PR-B mRNA increased approximately 8-fold in LUS and approximately 10-fold in the fundal myometrium of women in labor as compared with those not in labor (Fig. 2AGo). PR-A mRNA levels were low in LUS and fundal myometrium and were unaffected during labor (data not shown). By contrast, PR-C mRNA was dramatically up-regulated in fundal myometrial tissues of women in labor as compared with those not in labor. Thus, PR-B mRNA is induced in LUS and fundal myometrium of women in labor as compared with those not in labor, whereas PR-B protein is selectively increased in the fundus with labor. The fact that PR-B mRNA fails to be translated in the LUS during labor suggests a posttranscriptional regulatory event, such as decreased mRNA stability or a posttranslational event involving decreased protein stability (27). On the other hand, both PR-C mRNA and protein expression are spatially regulated in a coordinate manner in human myometrial tissue from women in labor. Q-PCR data indicate that PR-C isoform expression was increased greater than 200-fold in the fundal myometrium during labor. PR-C mRNA levels were undetectable in the LUS myometrium of women in labor but were increased to 4.68 ± 0.88 arbitrary units during labor (Fig. 2BGo). As indicated in Materials and Methods, one of the in-labor subjects from whom matched fundal and LUS myometrial samples were obtained (samples 4 and 10, IL; Fig. 2Go) presented with chorioamnionitis. Another matched pair of fundal and LUS myometrial samples (samples 6 and 12, IL; Fig. 2Go) were obtained from a subject at the time of hysterectomy immediately after vaginal delivery. Although it is possible that some of the changes in PR isoform expression could have been impacted by these clinical complications, it is evident that these changes occurred selectively in the fundal myometrium and therefore were associated with increased uterine contractility.

Selective Up-Regulation of PR-B and PR-C Expression in Fundal Myometrium from Women in Labor Is Associated with Activation of NF-{kappa}B
We previously observed that NF-{kappa}B is activated in mouse uterus (7) and baboon fundal myometrium (28) as term approaches. In the present study, we evaluated NF-{kappa}B p65 levels in cytoplasmic and nuclear fractions of LUS and fundal myometrium of women before and after the initiation of labor to determine whether changes in PR isoform expression in the fundus during labor were associated with activation of NF-{kappa}B. As can be seen in the immunoblots in Fig. 3Go, in LUS myometrium from women in labor and not in labor cytoplasmic levels of p65 were markedly elevated as compared with those in nuclear fractions, indicative of an absence of NF-{kappa}B activation. This was also the case for fundal myometrium from women not in labor. By contrast, in fundal myometrium from women in labor we observed a pronounced increase in nuclear levels of p65 relative to those in the cytoplasm. It should be noted that although one of the matched fundal and LUS myometrial biopsies (samples 4 and 10) was from a woman in labor presenting with chorioamnionitis, NF-{kappa}B was solely activated in fundal myometrium, suggesting that the presence of chorioamnionitis did not have a global impact on NF-{kappa}B activation within the uterus. Collectively, these findings suggest that NF-{kappa}B activation, which is restricted to fundal myometrium of women in labor, may play a role in the increase in PR-B and PR-C isoform expression observed (Fig. 2Go, A and B).


Figure 3
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  		Fig. 3. NF-{kappa}B Is Selectively Activated in Fundal Myometrium of Women in Labor

Nuclear and cytoplasmic extracts isolated from LUS and fundal myometrium of women in labor and not in labor (n = 3 for each group) were analyzed for levels of p65 expression by immunoblotting.

 
Expression of PR-A, PR-B, and PR-C Increase in the Pregnant Mouse Uterus in Late Gestation
As is shown in Fig. 4Go, protein and mRNA levels of PR-B, PR-A, and PR-C are up-regulated in the pregnant mouse uterus during late gestation, reaching maximal levels toward term. By immunoblotting, we observed that similar to fundal myometrium from women in-labor, PR-B levels in pregnant mouse uterus also paradoxically increase toward term (Fig. 4AGo). Q-PCR analysis of mRNA levels indicated a 9-fold increase in PR-B mRNA levels between 15 and 19 days post coitum (dpc) (Fig. 4BGo). PR-A protein was undetectable in mouse uterus from 15–18 dpc but was clearly evident in the nuclear fraction at 19 dpc (Fig. 4AGo). PR-A mRNA levels were relatively low through 17 dpc, increased approximately 5-fold on 18 dpc and further to approximately 9-fold at 19 dpc (Fig. 4CGo). Interestingly, mouse uterine PR-C protein was primarily cytoplasmic on 15–16 dpc and translocated to the nucleus on 17 dpc with nuclear levels increasing to 19 dpc (Fig. 4AGo). Although PR-C mRNA levels appeared to be increased at 18–19 dpc as compared with earlier time points, this increase was not found to be statistically significant (Fig. 4DGo).

Up-Regulation of Mouse Uterine PR Expression Correlates with Activation of the NF-{kappa}B Response Pathway in Vivo
We previously observed that NF-{kappa}B is activated in the pregnant mouse uterus toward term in association with the developmental induction of SP-A expression in fetal lung and its secretion into amniotic fluid (7). Furthermore, injection of SP-A into all amniotic sacs of the right uterine horn of 15 dpc pregnant mice caused preterm labor associated with a rapid (4.5 h) increase in fetal macrophage invasion and NF-{kappa}B activation in the injected uterine horn (7). Using the same intraamniotic injection protocol, we found that inhibition of NF-{kappa}B activation by intraamniotic injection of the NF-{kappa}B inhibitor SN50 caused a delay in the onset of labor (7). In the present study, we used this mouse model to determine whether the SP-A-induced macrophage invasion and NF-{kappa}B activation in the injected uterine horn was associated with increased expression of PR-B and PR-C isoforms. We also used this model to analyze changes in PR isoform expression with NF-{kappa}B inactivation. PR-B and PR-C protein levels were analyzed in the injected and noninjected contralateral uterine horns (4.5 h after injection of SP-A and 48 h after injection of SN50) by immunoblotting. As a control, a 15-dpc pregnant mouse was intraamniotically injected in the same manner with 3 µg protein from an SP-A-depleted extract or with a mutated and inactive form of the inhibitory SN50 peptide (SN50 mut) (7). As can be seen in Fig. 5AGo, nuclear levels of PR-B and cytoplasmic levels of PR-C proteins were increased in the SP-A-injected uterine horn as compared with the noninjected horn. No changes in PR-B and PR-C expression were observed in the uterine horn of a mouse injected with the SP-A depleted preparation (Fig. 5AGo, control). In contrast as can be seen in Fig. 5BGo, nuclear levels of PR-B and cytoplasmic levels of PR-C were decreased in the SN50-injected uterine horn, as compared with the noninjected and SN50 mut-injected uterine horns (Fig. 5BGo, control). Thus, it seems that increased expression of PR-B and PR-C isoforms are closely associated with the inflammatory response and NF-{kappa}B activation in the pregnant uterus.


Figure 5
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  		Fig. 5. PR-B and PR-C Expression Are Up-Regulated in Response to Activation of the NF-{kappa}B Pathway in Vivo

A, Three pregnant mice at 15 dpc were intraamniotically injected with SP-A or a mannose D-extracted, SP-A depleted preparation (Control) into the right uterine horn. SP-A injection results in an activation of NF-{kappa}B and the onset of labor in the injected uterine horn (7 ). Levels of PR isoform expression in the SP-A injected uterine horn vs. the noninjected and control-injected uterine horns (n = 3 for each group) were analyzed by immunoblotting 4.5 h after injection. B, Three pregnant mice at 15 dpc were intraamniotically injected with SN50 or SN50 mut (Control) into the right uterine horn. SN50 injection was found to block NF-{kappa}B activation and cause a delay in the onset of labor in the injected uterine horn, whereas SN50mut had no effect (7 ). Levels of PR isoform expression in the SN50-injected uterine horn vs. the noninjected and control-injected uterine horns (n = 3 for each group) were analyzed by immunoblotting 48 h after injection.

 
Activation of the NF-{kappa}B Response Pathway Increases PR Expression in Vitro
To further evaluate the role of NF-{kappa}B in the regulation of PR expression, we analyzed T47D human breast cancer cells, which express PR-A, -B, and -C isoforms, and hTERT myometrial cells, which express only PR-B, cultured for 12 h with IL-1ß (1, 5, 10, and 20 ng/ml), or SN50 (50 µg/ml). Nuclear levels of p65 were analyzed to assess NF-{kappa}B activation. As expected, levels of p65 were increased in a dose-dependent manner in the nuclear fractions of T47D (Fig. 6AGo) and hTERT (Fig. 6BGo) cells incubated with IL-1ß, as compared with controls. On the other hand, SN50 treatment caused a decrease in nuclear levels of p65 in both cell types. Interestingly, a corresponding up-regulation of PR-A, -B, and -C levels was seen in the IL-1ß-treated T47D cells and a down-regulation of PR isoform expression was observed in the SN50-treated cells (Fig. 6AGo). Using hTERT cells, we again found that IL-1ß-mediated activation of NF-{kappa}B was associated with a concomitant increase in PR-B expression. In contrast, cells treated with SN50 manifested a dramatic decline in PR-B isoform expression (Fig. 6BGo).

Activation of the NF-{kappa}B Response Pathway Increases p65 Binding to the hPR Promoter
To further define the mechanisms for IL-1ß induction of PR expression, we analyzed the 5' flanking sequence of the hPR gene (NM 000926) and identified a putative NF-{kappa}B response element at –1330 to –1319 bp (GGAAACTTTCC; NF-{kappa}B consensus = GGAAATTTCC). Chromatin immunoprecipitation (ChIP) analysis for NF-{kappa}B p65 binding to a 100-bp region containing this element was carried out in hTERT myometrial and T47D breast cancer cells cultured in the absence or presence of IL-1ß. As can be seen in Fig. 7Go, after 12 h of IL-1ß treatment, there was a pronounced increase in the in vivo binding of p65 to this region of the hPR promoter in both cell types. This suggests that cytokine-induced activation of NF-{kappa}B leads to increased p65 recruitment to the hPR promoter, resulting in an up-regulation of hPR expression.


Figure 7
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  		Fig. 7. IL-1ß Enhances Recruitment of NF-{kappa}B p65 to the hPR Gene Promoter

hTERT (A) and T47D (B) cells cultured for 12 h in serum-free medium with (hatched bar) or without IL-1ß (10 ng/ml) (white bar). 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. A no-antibody immunoprecipitation was used as a control (No Ab) (black bar). DNA was purified and the relative abundance of a 100-bp region surrounding the NF-{kappa}B response element 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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The persistence of elevated circulating levels of progesterone in the human maternal circulation throughout the third trimester of pregnancy and into labor has led us to postulate that labor is caused by a series of molecular events that impair PR function to maintain uterine quiescence. In most mammalian species, a progesterone withdrawal is associated with the initiation of parturition. In the pregnant mouse, a decline in progesterone levels precedes the onset of parturition and progesterone administration can delay the process (3). However, a role for progesterone withdrawal in the control of human labor has not been elucidated because circulating progesterone levels fail to decline before or during labor (4). Nevertheless, the fact that the PR antagonist RU486 can enhance cervical ripening and initiate labor (5) suggests that some form of progesterone deprivation might be involved in the initiation of labor in women. A functional progesterone withdrawal in human pregnancy could be mediated by a variety of mechanisms, including impairment of PR transcriptional activity by alterations in the levels of essential coregulators (6) and local activation of uterine inflammatory pathways (7).

Herein, we provide evidence that expression of the PR-C isoform, which inhibits endogenous PR-B transcriptional activity in cultured myometrial cells, increases markedly at term in both the pregnant mouse uterus and pregnant human uterus. Furthermore, our findings suggest that up-regulation of uterine PR expression is mediated by the activation of inflammatory response pathways that occurs in the pregnant uterus at term. Similar changes in PR isoform expression were observed in the pregnant mouse and human uterus during late gestation and into labor. In the pregnant mouse uterus, nuclear PR-B protein levels were found to increase modestly toward term, whereas PR-C levels increased in the cytoplasm between d 15 and 16 of gestation and then translocated to the nucleus where they continued to increase between 17 and 19 dpc (Fig. 4Go). PR-A, which was undetectable in cytoplasmic or nuclear fractions through 18 dpc, was only evident in the nucleus at 19 dpc. The increased levels of PR-A, -B, and-C proteins were associated with pronounced increases in the levels of mRNA for all three isoforms.

We also analyzed PR-A, PR-B, and PR-C protein and mRNA levels in fundal and lower uterine segment myometrium of pregnant women in labor and not in labor. As mentioned previously, studies on PR isoform expression to date have been carried out using LUS myometrium (23), which relaxes during parturition to allow expulsion of the fetus. However, we considered analysis of the fundus to be particularly important because this is the part of the uterus where contractility originates during labor (24) and therefore likely manifests withdrawal of PR function. As we observed in the mouse, PR-B mRNA levels increased dramatically in the fundal and LUS myometrial tissues of women in labor (Fig. 2AGo). On the other hand, a pronounced increase in PR-B protein levels was observed only in the laboring fundal myometrium. We suggest that the paradoxical increase in PR-B expression in both mouse and human uterus at term may be a consequence of an associated decrease in PR function. As mentioned above, we previously observed that expression of cAMP response element binding protein-binding protein and steroid receptor coactivators 1 and 2, as well as histone acetylation decreases in mouse and human myometrium at term (6). We postulate that the associated decrease in PR function may reduce the capacity of progesterone to negatively regulate PR expression (29), resulting in the up-regulation of a relatively inactive receptor.

Labor-associated changes in PR-C mRNA and protein levels in fundal myometrium were even more striking. PR-C mRNA was undetectable in the nonlaboring myometrium isolated from the LUS; however, PR-C mRNA levels increased to clearly detectable levels in LUS during labor (Fig. 2BGo). By contrast, an approximately 200-fold increase in PR-C mRNA levels was observed in the fundal myometrium of women in labor. PR-C protein, which was detectable only in cytoplasmic fractions, was undetectable in LUS but was markedly induced in fundal myometrium of women in labor (Fig. 2BGo). Although in the present study we were unable to detect nuclear or cytoplasmic PR-A protein in human fundal or LUS myometrium before or after the initiation of labor, PR-A protein has been reported to be up-regulated in total lysates isolated from the LUS myometrium of women in labor undergoing caesarean section as a result of fetal distress (22). Furthermore, the ratio of PR-A to PR-B mRNA was found to be increased 10-fold in term LUS myometrium from women in labor (23); however, in that study, the primers used for real-time RT-PCR would also have amplified PR-C mRNA. In total lysates of macaque myometrium, an increase in expression of PR-A relative to PR-B was observed in the myometrium at term; PR-C also was detected, although no discernible changes in relative levels of PR-C expression were found (30). It should be noted that, although PR-A has been proposed to serve as an antagonist of PR-B function in a cell- and promoter-specific manner (14) and was found to inhibit PR-B transcriptional activity in cultured human myometrial cells (22), mice with a selective knockout of the PR-B isoform are fertile and apparently deliver normally (17). This suggests that PR-A has the capacity to mediate the action of progesterone to maintain myometrial quiescence.

The cytoplasmic PR-C isoform has been suggested to inhibit PR-B action by sequestering locally available progesterone away from PR-B; when present in the nucleus, PR-C was observed to heterodimerize with PR-B and reduce its binding to response elements in DNA (20). PR-C, which lacks the first zinc finger of the DNA-binding domain and cannot bind to DNA, surprisingly was reported to enhance progestin-induced transcriptional activity of PR-B and PR-A isoforms (19). It was suggested that this enhancing action of PR-C may be due to the ability of PR-C to sequester corepressors away from PR-A and PR-B and/or to the increased capacity of heterodimers of PR-A or PR-B with PR-C to recruit coactivators (19). In contrast, we observed that PR-C caused pronounced inhibition of expression of an MMTV-luciferase reporter transfected into an immortalized myometrial cell line that expresses only endogenous PR-B (Fig. 1Go). Thus, it appears that PR-C, like PR-A, exerts an inhibitory effect on PR-B transcriptional activity in a cell- and promoter-specific manner. Furthermore, when this cotransfection assay was carried out in hTERT cells treated with IL-1ß to increase endogenous PR-B expression, PR-C still retained its capacity to inhibit PR-B transcriptional activity (Fig. 1Go). In addition as noted above, we suggest that, in the laboring mouse or human uterus, PR-B transactivating capability is already compromised due to decreased coactivator availability (6), increased levels of p65, which has already been demonstrated to inhibit PR transactivation (10). Therefore, based on these collective findings, it is likely that the relative expression levels of PR-A, -B, and -C proteins will determine the response of the myometrial cell to progesterone and that increased expression of the PR-C isoform may transform the uterine PR from a transcriptionally active state throughout most of pregnancy (maintaining uterine quiescence) to a repressed state at term (resulting in a loss of uterine quiescence).

Our findings further suggest that the labor-associated changes in PR isoform expression are associated with the activation of uterine inflammatory response pathways. NF-{kappa}B activation occurs in the pregnant mouse uterus between 17 and 19 dpc (7); this correlates with the observed changes in uterine PR isoform expression during this period (Fig. 4Go). We also found that intraamniotic injection of SP-A into 15 dpc mice, which promotes amniotic fluid macrophage migration to the maternal uterus, with increased cytokine production, uterine NF-{kappa}B activation, and preterm labor (7), caused a rapid increase in uterine levels of PR-B and PR-C proteins (Fig. 5AGo). In contrast, inhibition of uterine NF-{kappa}B activation by intraamniotic injection of SN50 (7) caused a decrease in the uterine levels of both PR-B and PR-C (Fig. 5BGo). In women, enhanced PR-B and PR-C expression in the fundal myometrium during labor also was correlated with NF-{kappa}B activation that was fundus-specific (Fig. 3Go). We previously reported that enhanced nuclear localization of NF-{kappa}B also occurred in fundal myometrium of the pregnant baboon during the latter third of gestation (28). In studies using T47D and hTERT myometrial cells as in vitro models, we found that the expression of all three PR isoforms was increased by activation of the NF-{kappa}B pathway in response to IL-1ß treatment. Furthermore, PR expression was decreased in these cells by inhibiting NF-{kappa}B activation (Fig. 6Go). Studies using ChIP analysis suggest that IL-1ß stimulates hPR expression through enhanced p65 binding to a putative NF-{kappa}B response element at –1330 bp upstream of the hPR gene (Fig. 7Go).

In the human uterus during pregnancy, there is clear spatial regulation of NF-{kappa}B activation (Fig. 3Go), coactivator (6), and PR isoform expression (Fig. 2Go). We suggest that this results in a selective increase in inflammatory response pathways and a decline in PR function within the uterine fundus. These changes are associated with a specific fundal increase in oxytocin receptors (31) and uterine contractility during labor. In recent studies, we also have observed that COX-2 mRNA levels are markedly increased in fundal myometrium of women in labor, whereas expression of COX-2 in LUS myometrium remains unchanged (32). This regionalization of the pregnant uterus is highlighted by studies using superfused human myometrial strips, in which prostaglandin E2 and F2{alpha} were found to stimulate contractility in the fundal myometrium, but not in the LUS (24). These observations are consistent with findings of Myatt and Lye (33), who reported increased expression of relaxant prostaglandin E2 receptors (EP4) and decreased expression of contractile prostaglandin F2{alpha} receptors (FP) in LUS myometrium with the onset of labor.

In conclusion, we have made the novel observation that activation of NF-{kappa}B within the mouse and human myometrium and the initiation of labor are associated with a striking increase in expression of the truncated inhibitory PR-C isoform. Because the marked increase in PR-C expression in mouse and human uterus at term occurs in association with a more modest increase in PR-B, we suggest the increase in PR-C:PR-B ratio may result in sequestration of cytoplasmic progesterone from PR-B and in the observed decrease in PR DNA-binding capacity in the laboring myometrium (34). The gestational increase in PR-C in the pregnant mouse uterus was prematurely induced after intraamniotic injection of SP-A into 15-dpc pregnant mice and associated with rapid activation of NF-{kappa}B and could be decreased by inactivation of the uterine NF-{kappa}B pathway. Cytokine treatment of T47D cells in culture also caused NF-{kappa}B activation, increased p65 binding to an NF-{kappa}B response element within the hPR promoter and increased PR isoform expression, whereas inhibition of NF-{kappa}B activation decreased PR expression both in vivo and in vitro. Finally, our findings in the human myometrium that NF-{kappa}B activation and PR-C expression are undetectable in the LUS and selectively up-regulated in the fundus at term provides a unique mechanism for spatial withdrawal of PR function leading to the initiation of labor-associated myometrial contractility within the fundus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Subjects and Tissue Acquisition
Fundal and lower uterine segment myometrial tissues were biopsied at term from pregnant women undergoing cesarean section, or at the time of hysterectomy immediately after vaginal delivery. Informed consent was obtained in writing from each woman before surgery using protocols approved by the Institutional Review Board of University of Texas Southwestern Medical Center in accordance with the Donors Anatomical Gift Act of the State of Texas. Myometrial biopsies were collected from two groups of subjects: 1) pregnant women who underwent cesarean section before the onset of labor at 35 wk gestation with no evidence of infection (cervical dilation was <3 cm); 2) pregnant women in active labor who underwent cesarean section at 39 wk gestation, or at the time of hysterectomy after vaginal delivery at 41 wk gestation (100 µg misoprostol was administered orally 7 h before delivery). One in-labor subject presented with evidence of chorioamnionitis. Cervical dilation in the in-labor group ranged from 2–9 cm. In groups 1 and 2, fundal and lower uterine segment myometrial biopsies were obtained by dissecting a strip of myometrium from the uterine wall opposing the site of implantation. All fundal (n = 3) and lower uterine segment (n = 3) biopsies from women in labor were obtained as matched pairs. Blood vessels were dissected from each of the biopsies, the myometrial smooth muscle tissues were cut into appropriate size pieces for subsequent immunohistochemical analyses and for flash freezing in liquid nitrogen and stored at –80 C. Myometrial smooth muscle was dissected from each of the biopsy samples and cut into appropriate size pieces, flash frozen in liquid nitrogen and stored at –80 C for subsequent protein and mRNA analysis.

Animal Surgery for Injection of Substances into Amniotic Fluid
All animal studies were approved by the Institutional Animal Care and Use Committee of University of Texas Southwestern Medical Center. Eight-week-old female ICR (an outbred Institute for Cancer Research strain) mice were housed with ICR male mice overnight. Mice found to have vaginal plugs at 0800 h were considered to be 0.5 dpc. Uterine tissues (n = 3 at each time point) were isolated from pregnant mice at 15–19 dpc. The uterine horn was cleared of all embryonic material and maternal decidua. The remaining whole uterine tissue was washed in 1x PBS and flash frozen for subsequent protein and mRNA analysis.

In studies to analyze the effect of NF-{kappa}B activation on uterine PR isoform expression, timed-pregnant ICR mice (n = 8) at 15 dpc were anesthetized with Avertin (2,2,2 tribromoethanol, tert isoamyl alcohol; Sigma-Aldrich, St. Louis, MO). The right uterine horn was gently pulled through a 1-cm incision made above the visible ovarian fat pad and substances [SN50, 10 pg/sac (Calbiochem, San Diego, CA) and SP-A, 3 µg/sac] in 50 µl were injected through the exposed uterine wall into all amniotic sacs with a sterile 31-gauge half-inch needle (Becton Dickinson, Franklin Lakes, NJ). As a control, 15-dpc pregnant mice were intraamniotically injected in the same manner with 3 µg protein from an SP-A-depleted extract (7) or 10 pg of a noninhibitory mutated form of SN50 (SN50 mut) (Calbiochem). The left uterine horn was untouched. The right uterine horn was returned to the abdominal cavity, the abdominal muscle wall was closed with ETHICON, 5–0 Chromic Gut sutures (Becton Dickinson), and the skin was closed using 9-mm wound clips (AUTOCLIP, Becton Dickinson). After injection, the mice were kept on a warm pad and returned to cages after recovery. The mice were then killed 4.5 h after injection of SP-A and 48 h after injection of SN50. The uterine horns were collected, cleared of all embryonic material and maternal decidua. The remaining whole uterine tissue was washed in 1x PBS and flash frozen for subsequent protein and mRNA analysis.

ChIP
hTERT and T47D cells were cultured for 12 h in serum-free medium, with or without IL-1ß (10 ng/ml). ChIP was performed using a modification of previously published methods (35). Briefly, cells (1 x 107 cells/treatment) (n =3) were washed once with PBS and then 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 1x PBS and placed in 500 µl 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 from three dishes were combined and sonicated on ice to produce sheared, soluble chromatin. The soluble chromatin was precleared with Protein A/G Plus agarose beads (60 µl) (Upstate Biotechnology, Lake Placid, NY) at 4 C for 1 h. The samples were then centrifuged at 14,000 rpm to pellet the beads, and the supernatant was placed in new tubes. The precleared chromatin was then aliquoted into 300-µl amounts and incubated with antibodies for p65 (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 C overnight. Two aliquots were reserved as controls—one incubated without antibody and the other with nonimmune IgG. Protein A/G Plus agarose beads (60 µl) were then 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 then 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 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 then 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, Carlsbad, CA) was then 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 for overnight at –20 C. Samples and input controls were diluted in 10–100 µl Tris-EDTA buffer just before PCR. Real-time PCR was carried out using primers (Table 1Go) that amplify an approximately 100-bp region containing the putative proximal NF-{kappa}B response element (ggaaactttcc) [consensus NF-{kappa}B response element is gggaaatttcc at position –1319 bp to –1330 bp of the hPR promoter (NM_000926)]. Using serial dilutions of human chromosomal DNA, these primers were demonstrated to have equal efficiency in priming their target sequences.


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  		Table 1. Primers Used in the Q-RT-PCR Analysis of Human Myometrial Biopsies and Mouse Uterus

 
Q-PCR
Total RNA from matched fundal and lower uterine segment myometrial tissues of six women in labor and six women not in labor and from three series of pregnant mice at 15–19 dpc was extracted by the one-step method of Chomczynski and Sacchi (36) (TRIzol; Invitrogen). RNA was treated with deoxyribonuclease to remove any contaminating DNA, and 4 µg were reverse-transcribed using random primers and Superscript II RNase H-reverse transcriptase (Invitrogen). The relative abundance of each PR isoform in both the mouse uterus and human myometrium was determined by real-time Q-PCR using a modification of previously published methods (23). Three primer sets directed against different regions of both the human and mouse PR were generated using Primer Express software (PE Applied Biosystems, Foster City, CA) based on published sequences (Table 1Go). The first human primer set, termed PR-B (37), was directed at the sequence specific for PR-B (upstream of the second ATG transcription initiation site), whereas the second human primer set, PR-AB, was directed at the sequence downstream of the second ATG translation initiation site. Finally, the human primer set for PR-ABC (38) was designed to the ligand-binding domain of the PR, a region common to all PR isoforms. A similar strategy was employed to generate mouse specific PR-B, PR-AB, and PR-ABC primers (Table 1Go). None of these primer sets corresponded to sequence in any of the other steroid hormone receptors. For the quantitative analysis of mRNA expression, the ABI Prism 7700 Detection System (Applied Biosystems) 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 where 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 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin as the reference guide for both the human and mouse, respectively. Over a wide range of known cDNA concentrations, both human and mouse PR 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 PR primer sets had equal priming efficiency, the {Delta}Ct values (PR primer-internal control) for each PR primer set were calibrated to the uterine samples with the lowest PR abundance (highest Ct value) and the relative abundance of each primer set compared with calibrator was determined by the formula, 2–{Delta}{Delta}Ct, whereby {Delta}{Delta}Ct is the calibrated Ct value. The relative abundance of PR-A could then be calculated by subtracting the relative abundance of PR-B from that of PR-AB, whereas the relative amount of PR-C was calculated by subtracting the relative abundance of PR-AB from PR-ABC.

Immunoblot Analysis
Human myometrial and mouse uterine tissue cytoplasmic extracts were prepared by homogenizing the tissue 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 mM 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, the supernatant was retained as the nuclear fraction (26). Equal concentrations of nuclear and cytoplasmic proteins, normalized by colorimetric bicinchoninic acid protein assay (Pierce, Rockford, IL), were fractionated in gradient polyacrylamide gels (Invitrogen) and transferred onto Hybond-P (Amersham Pharmacia, Arlington Heights, IL). Blots were probed using rabbit antibodies for PR (C-19): sc-538 (Santa Cruz Biotechnology) (1:500), which recognizes PR-A, PR-B, and PR-C isoforms, and NF-{kappa}B p65 (C-20): sc-372 (Santa Cruz Biotechnology) 1:200 diluted in 5% milk-1x TBS-T buffer and with horseradish peroxidase-conjugated goat antirabbit IgG (1:10,000) diluted in 5% milk-1x TBS-T buffer (Amersham Pharmacia) as the secondary antibody. Immunoreactive bands were visualized using an ECL-detection system (Amersham Pharmacia).

Cell Lines
Human breast cancer T47D cells were maintained in RPMI medium with 10% FBS and 0.1 mM insulin. Cells were treated with SN50 (39) (50 µg/ml) (Calbiochem), progesterone (100 nM) (Sigma) or IL-1ß (1, 5, 10, and 20 ng/ml) (Sigma) for 12 h. Human myometrial hTERT cells (25) were maintained in DMEM-F12 (Invitrogen) with 10% FBS. Cells were treated with SN50 (50 µg/ml), IL-1ß (1, 5, 10, 20 ng/ml), for 12 h. Cell lysates and nuclear extracts were prepared as described previously (26) and analyzed by immunoblotting.

hPR-C Subcloning
hPR-C was amplified by PCR using forward (5'-CAC CAT GGA AGG GCA GCA CAA C-3') and reverse (5'-TCA TCA CTT TTT ATG AAA GAG AGG-3') primers from a PR-B-containing plasmid (pBKCMV-hPR-B) (11) and subcloned into pcDNA 3.1 Topo (Invitrogen) expression vector.

hTERT Transient Transfection
Telomerase-immortalized human myometrial cells (hTERT) (25) were cultured in DMEM-F12 medium supplemented with 10% FBS. Cell monolayers were subcultured onto 12-well culture dishes in 1 ml DMEM/F12 medium (Invitrogen) and transfected 24 h later. Transfection was performed with the indicated concentrations of the hPR-C isoform expression plasmid (CMV:PR-C) or empty vector (EV) (pcDNA 3.1 Topo) and a progesterone-responsive reporter plasmid comprised of the mouse mammary tumor virus LTR fused to luciferase (LUC), as reporter (MMTV-Luc) (14) (1 µg) with 2 µl of Fugene 6 (Roche) per 1 µg of plasmid DNA for 6 h at 37 C. The total amount of plasmid DNA transfected was kept constant by addition of empty pcDNA 3.1. After 6 h of transfection, cells were then incubated with 1.0 ml low-serum medium overnight at 37 C. After overnight recovery, cells were treated with progesterone (100 nM) and IL-1ß (10 ng/ml), and the PR antagonist RU486 (100 nM) for 12 h, lysed and assayed for reporter activity using a dual luciferase reporter assay system (Promega). Results are expressed as a percentage of basal luciferase activity (values were normalized for transfection efficiency using a cotransfected Renilla luciferase vector and total protein concentration by colorimetric bicinchoninic acid protein assay. Values represent the mean ± SEM of determinations from three independent experiments, each performed in triplicate.


   FOOTNOTES
 
This research was supported by National Institutes of Health Grant 5 P01 HD011149 and a research grant from the March of Dimes Birth Defects Foundation No. 21-FY04-174 obtained by C.R.M.

The authors J.C.C., D.B.H., K.K. and C.R.M. have nothing to declare.

First Published Online December 8, 2005

Abbreviations: ChIP, Chromatin immunoprecipitation; COX-2, cyclooxygenase-2; Ct, comparative cycle time; dpc, days post coitum; hPR, human PR; hTERT, human telomerase reverse transcriptase; ICR, an outbred Institute for Cancer Research strain; LUS, lower uterine segment; MMTV, mouse mammary tumor virus; NF, nuclear factor; Q-PCR, quantitative PCR; PR, progesterone receptor; PR-A, -B, and -C, PR isoforms; SDS, sodium dodecyl sulfate; SP-A, surfactant protein A.

Received for publication June 20, 2005. Accepted for publication November 28, 2005.


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

Nuclear Receptors:   PR
Ligands:   Progesterone  |  RU486



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