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BOR - Papers in Press, published online ahead of print December 11, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.011684
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BIOLOGY OF REPRODUCTION 68, 1687–1694 (2003)
DOI: 10.1095/biolreprod.102.011684
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Decidual Activin: Its Role In the Apoptotic Process and Its Regulation by Prolactin1

Christian Tessier3,7, Anne Prigent-Tessier4,7, Lei Bao7, Carlos M. Telleria5,7, Susan Ferguson-Gottschall7, Gil B. Gibori7, Yan Gu6,7, Jennifer M. Bowen-Shauver7, Nelson D. Horseman8, and Geula Gibori2,7

Department of Physiology and Biophysics,7 University of Illinois, Chicago, Illinois 60612-7432 Department of Molecular and Cellular Physiology,8 University of Cincinnati, Cincinnati, Ohio 45267-0576


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Successful pregnancy requires profound differentiation and reorganization of the uterine tissues including, as pregnancy progresses, extensive apoptosis of decidual tissue to accommodate the developing conceptus. We have previously shown a positive correlation between expression of activin A and apoptosis in the decidua and have also shown that expression of activin A occurs at the time when prolactin (PRL) receptors disappear from decidual cells. The goals of this study were to examine whether activin A plays a role in decidual apoptosis and whether expression of activin A in the decidua is regulated by PRL and placental lactogens. Studies were carried out using primary rat decidual cells, a decidual cell line (GG-AD), and PRL null mice. Treatment of decidual cells with activin A significantly increased DNA degradation, caspase 3 activity, and caspase 3 mRNA expression. However, this effect was observed only in the absence of endogenous activin production by these cells. Addition of follistatin to decidual cells that were producing activin A decreased both caspase 3 activity and mRNA expression. Similarly, addition of activin-blocking antibodies to cultures of GG-AD cells, which also produce activin A, caused a reduction in both DNA degradation and caspase 3 activity. PRL and placental lactogens caused an inhibition of activin A mRNA expression in primary decidual cells. Even more convincingly, decidua of PRL null mice expressed abundant activin A at a time when no expression of this hormone is detected in wild-type mice and treatment of PRL null mice with PRL caused a profound inhibition of activin A mRNA expression. In summary, our investigations into the role and regulation of decidual activin have revealed that activin A can induce cell death in the decidua and that its expression is under tight regulation by PRL and placental lactogens.

activin, apoptosis, decidua, prolactin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The formation of the decidua involves not only proliferation and differentiation of endometrial stromal cells, but also extensive regression and reorganization of the decidual tissue, including cell death by apoptosis [1–4]. To accommodate the developing conceptus, a great number of decidual cells must die. This cell death does not occur at random. Cell death begins in the antimesometrial region, which becomes ultimately reduced to a thin layer of cells (the decidua capsularis in pregnant rodents, or antimesometrial decidua in pseudopregnant rodents), and later extends to the mesometrial cells, forming the decidua basalis (or mesometrial decidua), which is in close contact with the placenta. Although cell death is extensive and increases with time, it is always more pronounced in the antimesometrial tissue than in the mesometrial [1, 2]. Despite the importance of decidual regression for the success of pregnancy, what causes apoptosis to occur in decidual cells and what controls its pattern of expression is still not known. The decidual tissue, interestingly, regresses with similar morphology and kinetics whether decidual development was induced by the implanting blastocyst or by artificial means [5, 6], suggesting that the signals that either prevent or induce decidual cell death do not emanate from the conceptus but are produced by the decidua itself.

The decidua expresses a number of hormones and cytokines that could be involved in the regulation of decidual apoptosis. Among these is activin A. We [7] and others [8] have previously shown that in rats and mice the decidua abundantly expresses activin A, a dimer of the ßA inhibin subunit. Neither the ßB subunit nor {alpha}-inhibin could be detected in the decidua, indicating that activin A is the only member of this group of hormones produced in this tissue. It is interesting that the activin A gene is expressed early and transiently in the "primary decidual zone" that forms and then rapidly disappears just after implantation. The activin A gene is silenced thereafter in the decidua, but it becomes abruptly expressed after Day 11 of pregnancy, coincident with the onset of apoptosis in decidual cells [7]. Further, the level of expression of the activin A gene is much higher in the antimesometrial decidua (where apoptosis is more extensive) than in the mesometrial decidua [1].

The successful knock out of the activin A gene [9] did not help to define the local role of decidual activin A because mice deficient in activin A die at birth. However, the knockout approach did establish an important apoptotic role of activin in the fetal liver [10]. Thereafter, activin was also shown to induce apoptosis in many adult tissues and cell lines [11–15]. The abundant evidence that activin can act as a proapoptotic factor, coupled with the tight correlation that exists between the expression of activin A and decidual apoptosis, suggests a possible apoptotic role for activin A in the decidua.

In the last decade, numerous genes have been linked to apoptosis. However, recent investigations have revealed that, ultimately, cell death is caused by a set of cysteine proteases, named caspases, which are specifically activated in apoptotic cells. Most of the morphological changes observed during apoptosis, including chromatin condensation and fragmentation, membrane blebbing, and ultimate break up, are caused by cellular caspases [16–18]. Among the caspases, caspase 3 appears to be the key protease in the apoptotic pathway [16] and is responsible for DNA fragmentation. We have recently shown [19] that active caspase 3 becomes detectable in the decidua, coincident with the expression of activin A and the onset of apoptosis. However, whether there is a causal link between activin A and activation of caspase 3 is unknown. In fact, neither the role of decidual activin A, nor the factor or factors that control its expression during development are known.

Our previous finding that activin A becomes expressed in the decidua at the time when the prolactin (PRL) receptor (PRL-R) disappears from this tissue [7, 20] suggested to us that PRL secreted by the rat decidua [21], or PRL-like hormones secreted by the trophoblast (rPL-1 and rPL-2) may inhibit decidual activin A expression. The loss of PRL-R at defined times during decidual development could then serve as a trigger to permit activin A expression. Recent evidence obtained from PRL-R and PRL null mice has revealed that induction of decidualization [22] and implantation [22, 23] occur normally in these mice if they are treated with progesterone to compensate for the luteal defect. However, despite progesterone treatment, fetal death starts at midpregnancy [22, 23]. It is possible that this may be the result of premature expression of apoptotic factors, perhaps including activin A, and thus early decidual apoptosis. Using primary rat decidual cells, a rat decidual cell line and PRL knockout mice, the goal of this study was to examine whether activin A plays a role in the apoptotic process in the decidua and whether PRL or the PRL-like hormones produced by the trophoblast regulate its expression in that tissue.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Acrylamide and bis-acrylamide were obtained from Accurate Chemical Inc. (Westbury, NY) and Eastman Kodak (Rochester, NY), respectively; Takara Ex Taq DNA polymerase was purchased from Pan Vera Corporation (Madison, WI), [32P]-deoxycytidine triphosphate {alpha} ([32P]dCTP) was from Amersham (Arlington Heights, IL), the oligonucleotides used as primers in the reverse transcription-polymerase chain reaction (RT-PCR) analysis were obtained from Life Technologies Inc. (Grand Island, NY). Tissue culture medium (RPMI-1640), antibiotic-antimycotic solution, nonessential amino acids, and sodium pyruvate were from Mediatech (Washington, DC); fetal bovine serum (FBS) was purchased from Hyclone Laboratories Inc. (Logan, UT); progesterone and all other reagent grade chemicals were purchased from Sigma Chemical Co. (St. Louis, MO); ovine PRL (oPRL, PRL-18, 30 IU/mg) and recombinant human activin A were a gift from the National Institute of Diabetes and Digestive and Kidney diseases (Bethesda, MD). Rat placental-lactogen I (rPL-I) and II (rPL-II) were kindly provided by Dr. Robert Shiu (University of Manitoba, Winnipeg, Manitoba, Canada).

Animals

Mice with germ line transmission of the PRL null mutation [24] were kept at 25°C with a 14L:10D cycle and were fed a commercial pelleted diet ad libitum. Heterozygous mutants in the C57BL/6 x 129/sv background were intercrossed to generate +/+, +/-, and -/- (null) mice, which were genotyped by PCR amplification using tail DNA, as previously described [24]. Pseudopregnant rats of the Holtzman strain were obtained from Harlan (Madison, WI). Rats were kept under controlled conditions of light (14L:10D, lights-on 0500–1900 h) and temperature (22–24°C) with free access to standard rat chow and water. Pseudopregnancy was induced in both rats and mice by mating with vasectomized males and the day on which a vaginal plug was found was designated Day 1 of pseudopregnancy. Decidualization of uterine endometrium was induced in rats by scratching the antimesometrial surface of both uterine horns with a hooked needle on Day 5 of pseudopregnancy under ether anesthesia. Decidualization in mice was induced on Day 4 of pseudopregnancy by injecting sesame oil (25 µl) intraluminally in each uterine horn under ether anesthesia. Decidualization was sustained in PRL null mice by injections of progesterone (3 mg s.c. in sesame oil, once daily), prolactin (60 µg s.c. in polyvinyl pyrrolidone, twice daily), or progesterone plus prolactin beginning on the day of vaginal plug detection. All experimental procedures were performed in accordance with the principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.

Primary Cell Culture

Decidual cells in primary culture were collected from decidual tissue on Day 9 of pseudopregnancy as previously described [21]. Cells (1.2–1.5 x 106) were seeded in 6-well plates and incubated at 37°C in a 95% air/5% CO2 humidified atmosphere in RPMI-1640 medium containing 2x antibiotic-antimycotic solution (200 U/ml penicillin G, 0.5 µg/ml amphotericin B, and 200 µg/ml streptomycin), 1x nonessential amino acids, 1 mM sodium pyruvate, 0.45% D-glucose, and 10% FBS. Cells were allowed to attach for 3–4 h, washed, and then cultured for 12 to 72 h in RPMI-1640 phenol red-free medium supplemented with 1% charcoal-dextran-stripped FBS with or without treatment. At the end of the experiment, cells were washed twice with ice-cold PBS and stored at -80°C until extraction of RNA. For DNA fragmentation or caspase 3 activity analysis, both culture medium containing detached cells and attached cells were frozen at -80°C.

GG-AD Cell Culture

The temperature-sensitive GG-AD cells derived from rat antimesometrial decidual cells [25] were grown for 3 days at 39°C (the differentiating temperature) in RPMI-1640 medium supplemented with 2x antibiotic-antimycotic solution (200 U/ml penicillin G, 0.5 µg/ml amphotericine B, and 200 µg/ml streptomycin), 1x nonessential amino acids, 1 mM sodium pyruvate, and 0.45% D-glucose, and 1% charcoal-dextran-treated FBS in a 95% air/5% CO2 humidified atmosphere. The cells were then treated with activin A blocking antibody (1:100 and 1:25 dilutions) for 48 h at 39°C. At the end of the incubation, both culture media containing detached cells and attached cells were frozen at -80°C until DNA isolation.

DNA Fragmentation

The internucleosomal cleavage of DNA was analyzed for cultured primary decidual cells as follows. Both detached and attached cells were collected and centrifuged for 5 min at 1500 x g. The cell pellets were resuspended and incubated at 50°C overnight in 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 25 mM EDTA pH 8.0, 0.5% SDS, and 0.1 mg/ml proteinase K (Life Technologies Inc., Gaithersburg, MD). DNA from the digested cells was extracted with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v). The DNA was precipitated and digested for 1 h at 37°C in the presence of 1 µg/ml RNase (DNase free, Roche, Indianapolis, IN). After extraction and precipitation, an equal amount of DNA (3 to 5 µg) was separated by electrophoresis on a 1% agarose gel impregnated with ethidium bromide. The DNA pattern was examined via UV transillumination.

To examine DNA fragmentation in GG-AD cells, we used the 3'-labeling procedure previously described by Tilly et al. [26]. Briefly, the reaction solution consisted of sample DNA (0.5 µg) from attached and detached cells, reaction buffer, terminal transferase (25 units, Promega, Madison, WI) and 10 µCi of [32P]dCTP. The labeling reaction was carried out at 37°C for 1 h followed by 10 min at 70°C. DNA fragments were electrophoretically separated on a 2% agarose gel. After drying, the gel was examined by autoradiography.

Caspase 3 Activity

Caspase 3 activity was measured using the colorimetric ApoAlert caspase 3 assay kit (ClonTech Laboratories Inc., Palo Alto, CA) according to the manufacturer's instructions. Briefly, both detached and attached cells were collected from culture medium and centrifuged for 5 min at 1500 x g. Cell pellets were resuspended in PBS and an aliquot was kept for protein measurement using the bicinchoninic acid (BCA) kit (Pierce, Rockford, IL). After centrifugation, cell pellets were resuspended in ice-cold cell lysis buffer and were incubated on ice for 10 min. At the end of the incubation, cell lysates were centrifuged at 10 000 x g to precipitate cellular debris. Supernatants were then incubated for 1 h at 37°C in the presence of 1 mM caspase 3 substrate (DEVD-pNA) and the optical density was measured at 405 nm.

RNA Isolation and RT-PCR Analysis

Total RNA from decidual cells in primary culture was purified using TRI Reagent (Sigma), whereas total RNA from the decidual tissue of mice was isolated using Trizol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's instructions. The RT and PCR reactions were conducted as previously described [27]. For the PCR reaction, the conditions were such that amplification of the product was in the exponential phase, and the assay was linear with respect to the amount of input cDNA. Reaction products were electrophoresed on an 8% polyacrylamide nondenaturing gel. After autoradiography, data were quantified using a PhosphorImager and ImageQuant version 3 software (both from Molecular Dynamics, Sunnyvale, CA) and normalized to the expression of mRNA for L19 ribosomal protein. Oligonucleotide primer pairs used for mRNA analysis by RT-PCR, with annealing temperatures and predicted product sizes, are listed in Table 1.


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  		TABLE 1. Oligonucleotide primer pairs used for mRNA analysis

Statistical Analysis

Data were examined by one-way ANOVA followed by the Duncan or the Tukey multiple range test. When appropriate, the Student t-test was used. A level of P < 0.05 was accepted as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Activin A on Decidual Cell DNA Breakdown and Caspase 3 Activity

To determine whether activin A can induce apoptosis, we used both the GG-AD cells and primary decidual cells obtained from Day 9 pseudopregnant rats. The GG-AD cells are derived from SV-40 transformed rat decidual cells and are known to highly express activin A [25]. These cells were cultured at 39°C for 72 h to induce differentiation and were then incubated for an additional 48 h in the presence of 1:100 or 1:25 activin A blocking antibody or with normal serum (NS). As shown in Figure 1A (lanes 5 and 6), extensive DNA degradation took place in these cells under control conditions (NS only). Addition of activin A blocking antibodies caused a marked reduction in DNA degradation (Fig. 1A, lanes 1–4). Our previous finding that caspase 3 mRNA levels are highly expressed when decidual tissue undergoes apoptosis [19] led us to examine whether this caspase, which is known to cause DNA degradation [18], is stimulated by activin A. As shown in Figure 1B, caspase 3 was highly active in GG-AD cells cultured under control conditions, which were undergoing severe DNA breakdown, and addition of activin A blocking antibodies reduced the activity of this caspase.



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  		FIG. 1. Effect of activin A blocking antibodies on DNA fragmentation and caspase 3 activity in GG-AD cells. Differentiated GG-AD cells were incubated for 48 h in the presence of activin A blocking antibody. A) Representative DNA fragmentation gel. B) Caspase 3 activity, average of three separate experiments. Asterisks indicate P < 0.05 compared to normal serum treatment

We further examined the apoptotic effect of activin A using primary decidual cells obtained from Day 9 pseudopregnant rats. At this stage the decidua does not express activin in vivo [7]. Decidual cells were grown in the presence of increasing concentrations of activin A in media containing 1% charcoal-dextran-treated FBS. Genomic DNA and protein were isolated and both DNA fragmentation and caspase 3 activity were assessed after 12 h of culture. Treatment of primary decidual cells with activin A for 12 h significantly increased DNA degradation (Fig. 2A), and this was accompanied by a marked increase in caspase 3 activity (Fig. 2B). We found it interesting that neither DNA breakdown (Fig. 3A) nor caspase 3 activity (Fig. 3B) were increased by the addition of activin A when decidual cells were cultured for more than 12 h. We observed that decidual cells in culture express activin A and that this expression increases markedly after 24 h of culture (Fig. 3C), suggesting that endogenously produced activin A may activate caspase 3 and induce apoptosis at these later time points.



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  		FIG. 2. Activin A mediated stimulation of DNA fragmentation and caspase 3 activity in decidual cells. Cells were cultured for 12 h in the presence of activin A. A) Representative results for DNA fragmentation. B) Caspase 3 activity, average of three separate experiments. Asterisks indicate P < 0.05 compared to control (0 ng/ml)



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  		FIG. 3. Effects of activin A and follistatin on decidual cells in primary culture. Cells were cultured with 25 ng/ml activin A. A) DNA degradation after 12, 24, and 48 h of culture, representative results. B) Caspase 3 activity after 12 or 24 h of culture (n = 3 experiments). Asterisk indicates P < 0.05 compared with that of control at that time (t-test). C) Semiquantitative RT-PCR of total RNA from cells cultured for 12, 24, or 48 h. (n = 3). Asterisks indicate P < 0.05 compared with the 12-h time point. D) In separate experiments, decidual cells were cultured for 72 h in the presence of 100 ng/ml follistatin. Caspase 3 activity was measured as described in Materials and Methods (n = 3) and caspase 3 mRNA was measured by semiquantitative RT-PCR (n = 3). Asterisks indicate P < 0.05 compared to untreated cells

To examine this possibility, decidual cells were cultured in the presence of follistatin, which binds to activin and eliminates its activity [28]. Addition of follistatin to the culture markedly decreased the activity of caspase 3 (Fig. 3D). It was interesting to learn that addition of follistatin to the culture also led to a decrease in caspase 3 mRNA, suggesting a role for decidual activin A on caspase 3 expression. Indeed, when activin A was added to decidual culture (Fig. 4), it induced a robust stimulation in caspase 3 mRNA levels.



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  		FIG. 4. Effect of activin A on caspase 3 mRNA expression in decidual cells in primary culture. Decidual cells were cultured for 72 h with 100 ng/ml activin A. Caspase 3 mRNA was measured using semiquantitative RT-PCR. The left panel depicts one representative autoradiogram for three sets of mRNA and the right panel shows the normalized mRNA levels as mean ± SEM. (n >= 3). Asterisk indicates P < 0.05 compared to untreated cells

Regulation of Decidual Activin A by Prolactin and Placental Lactogens

Our previous finding that activin A becomes expressed in the decidua only at a time when PRL-R disappear from this tissue [20] led us to examine the effect of PRL treatment on activin A expression. Decidual cells in primary culture were grown for 12 h with different concentrations of PRL and the effect of these treatments on activin A mRNA levels was determined by semiquantitative RT-PCR. PRL at several doses caused an inhibition of activin A mRNA expression (Fig. 5). Rat placental lactogens I and II produced in the later stages of pregnancy by the trophoblast also bind to the decidual PRL-R and activate the same signal transduction machinery. Therefore, we examined the effect of rPL-I and rPL-II on activin A mRNA in the same culture system and found that both rPL-I (Fig. 6A) and rPL-II (Fig. 6B) could significantly down-regulate activin A mRNA expression.



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  		FIG. 5. PRL effect on activin A mRNA expression in decidual cells in primary culture. Decidual cells were cultured for 12 h in the presence of different doses of oPRL. Activin ßA mRNA was measured using semiquantitative RT-PCR One representative autoradiogram is shown in the upper panel. The densitometric analysis from n >= 3 independent experiments (mean ± SEM of the normalized mRNA levels) is depicted in the lower panel. Asterisks indicate P < 0.05 compared to untreated cells



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  		FIG. 6. Effect of rPL-I and rPL-II on activin A mRNA expression in decidual cells in primary culture. Decidual cells were cultured for 12 h in the presence of different doses of rPL-I or rPL-II. Activin ßA mRNA was measured using semiquantitative RT-PCR. A) Effect of rPL-I on activin A mRNA, (B) effect of rPL-II. One representative autoradiogram is shown in the upper panels. The lower panels depict the densitometric analysis of the normalized mRNA levels (mean ± SEM; n >3). Asterisks indicate P < 0.05 compared to untreated cells

To further investigate the effects of PRL on decidual expression of activin A we used pseudopregnant PRL null mice. These mice were injected daily from Day 1 of pseudopregnancy, the day following mating, with either PRL, progesterone, or a combination of both hormones. Treatment with progesterone or PRL is necessary to maintain pseudopregnancy and permit decidualization in these mice because it corrects the luteal deficiency. Wild-type mice served as controls. Decidualization of PRL null and wild-type mice was induced on Day 4 of pseudopregnancy and decidual tissue was isolated 6 days later. As shown in Figure 7, the decidua of wild-type mice does not express mRNA for activin A on Day 10 of pseudopregnancy. In contrast, the decidua of PRL null mice treated with progesterone alone expresses abundant activin A mRNA. Treatment of PRL null mice with PRL, alone or in concert with progesterone, resulted in the total silencing of activin A mRNA expression.



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  		FIG. 7. Effect of PRL and progesterone on activin A expression in decidua of PRL null mice. Induction of decidualization was carried out in wild-type and PRL null mice as described in Materials and Methods. PRL null mice were injected with progesterone (3 mg s.c. in sesame oil, once daily), PRL (60 µg sc in polyvinyl pyrrolidone, twice daily), or progesterone plus PRL beginning on the day of vaginal plug detection. Decidual tissues were collected from different groups of mice on Day 10 of pseudopregnancy, and mRNA for activin ßA was measured using semiquantitative RT-PCR. The mRNA levels are expressed in the lower panel as the mean ± SEM (n = 3). Asterisk indicates P < 0.05 compared to all other groups


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our investigations into the role and regulation of decidual activin have revealed that activin A can induce apoptosis in decidual cells and that its expression by these cells is inhibited by PRL and by PRL-like hormones secreted by the placenta. The finding that activin A blocking antibodies or an activin A binding protein can prevent DNA fragmentation and caspase 3 activation in decidual cells that produce endogenous activin A indicates that endogenously produced activin A can act as a proapoptotic factor in the rat decidua. Activin A has been shown to have a variety of biological activities [29], including the regulation of cell proliferation and the induction of apoptosis in mammalian cells. Cells in which activin A has been demonstrated to induce apoptosis include a human prostate cancer cell line [13], B-cells [14], and a human hepatoma cell line [30]. It is interesting that TGFß1, which belongs to the same growth factor family as activin A, has been shown to induce programmed cell death in endometrial stromal cells in primary culture [31]. These results taken together suggest an important role for the TGFß growth factor family in the remodeling of decidual tissue during pregnancy. The expression of activin A, rather than TGFß1, coincident with apoptosis in the rat decidua indicates that activin A is the physiological factor that stimulates apoptosis in this tissue.

Activin A carries out its biological role through two types of cell surface receptors, named type I and type II receptors [7, 8]. Following ligand stimulation, these receptors activate and phosphorylate the Smad family of proteins, which translocate to the nucleus and regulate transcription of target genes [32, 33]. The signal transduction pathway used by activin A to carry out apoptotic effects in the rat decidua remains to be investigated. However, both the type I and type II receptors, as well as the Smad proteins, have been shown to play an important role in activin-mediated apoptosis in B cells [14, 15, 34] and hepatocytes [30].

The caspase family of proteases plays a central role in apoptosis. Caspase 3 is synthesized as proenzyme and is proteolytically cleaved into an activated form. Active caspase 3 causes activation of the ladder nuclease (now known as caspase-activated DNase or CAD), which in turn causes DNA fragmentation. In nonapoptotic cells, CAD is present as an inactive complex with its inhibitor, ICAD. During apoptosis, ICAD is inactivated by caspase 3, leaving CAD free to enter the nucleus to produce DNA fragmentation [35, 36]. Deletion of the caspase 3 gene is lethal, and mice deficient in caspase 3 [37] show a specific defect in apoptosis that includes incomplete chromatin condensation and absence of DNA fragmentation. Also, cells that do not express caspase 3 (such as MCF-7 cells) do not undergo DNA fragmentation during cell death [35, 38]. Our study clearly demonstrates that one of the cellular targets of activin A is caspase 3. Indeed, activin A was able to up-regulate both caspase 3 activity and caspase 3 mRNA levels in decidual cells. These results may explain why mRNA for both activin A and caspase 3 appear together in the decidua at the time when this tissue undergoes extensive apoptosis [1, 7, 19]. Apoptosis induced by the closely related molecule, TGFß, has been shown to be dependent on caspase 3 stimulation in both B cells and hepatocytes [39–41]. However the mechanism by which these molecules lead to the activation of this caspase is not known.

Previous studies from our laboratory have shown a correlation between the disappearance of the PRL receptor from the decidua, the appearance of activin A mRNA, and apoptosis in this tissue [1, 7, 20]. PRL-R is expressed at constant levels in the rat decidua until midpregnancy, when its level of expression becomes reduced first in the antimesometrial and then in the mesometrial cells. Concomitant with the decrease and disappearance of the PRL-R is a rather abrupt expression of activin A; moreover, a similar inverse relationship between the PRL-R and activin A expression was observed in primary decidual cells in culture [20]. We have also recently described a role for PRL as an important survival factor in the rat decidua [19]. Whether all or part of the antiapoptotic effect of PRL in the decidua is due to its ability to inhibit activin A expression remains to be investigated. Nevertheless, our finding that activin A causes cell death in the decidua, together with the fact that it becomes highly and differently expressed in the antimesometrial and mesometrial decidua around midpregnancy, when extensive cell death is needed, suggest an important physiological role for activin A in the regression and reorganization of the decidual tissue. Suppression of activin A expression during early decidual development, therefore, may be important to prevent premature cell death. The factors that inhibit and direct activin A expression during decidual development are not known. Our findings that PRL and PRL-related hormone such as rPL-I and rPL-II inhibit activin A expression in decidual cells suggest that decidually derived PRL [21] and PRL-like hormones secreted by the trophoblast [42] may inhibit and direct activin A expression during decidual development.

Our results obtained with the PRL knockout mice further support an important role for decidual PRL in activin A expression and the normal progress of pregnancy. PRL and PRL-R null mice are infertile. Because the most important role of PRL in fertility is its ability to sustain the corpus luteum production of progesterone [43], these mice were treated with progesterone, which rescued implantation and allowed the normal progress of decidualization [22, 23]. However, extensive fetal death started from midpregnancy despite steroid treatment and no progesterone levels could entirely salvage normal fetal development. This led investigators to conclude that decidually produced PRL may play a key role in the normal progress of pregnancy, acting at the level of the decidua. Indeed, our previous investigations on the roles of PRL and PRL-R in rat decidua have revealed that PRL has potent inhibitory and stimulatory roles on some key decidual genes [19, 27, 44–47]. Of great interest to us is our present finding that PRL inhibited the decidual expression of activin A in the PRL null mice in a powerful manner. In sharp contrast, PRL null mice treated with progesterone expressed activin A abundantly. It is therefore highly possible that the premature abortion and fetal death seen in the PRL and PRL-R null mice treated with progesterone is due, at least in part, to high decidual expression of activin.

In summary, our investigations into the role and regulation of decidual activin have revealed that activin A can induce cell death in the decidua and that its expression is under tight regulation by PRL and placental lactogens. Further, we have determined that activin A carries out its proapoptotic effects at least in part by enhancing activation and expression of the executioner caspase, caspase 3.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Robert Shiu for providing the rat placental-lactogen I and II and to Dr. Michio Takahashi for the activin antibody. We also thank the National Institute for Diabetes and Digestive and Kidney Diseases and the National Hormone and Pituitary Program for providing us with oPRL and recombinant human activin A.


   FOOTNOTES
 
1 This work was supported by National Institutes of Health grants HD-12356, U54 HD 40093, and HD-11119 (to G.G.), by the Ernst Schering Research Foundation (to C.T.), and by NIH grant T32 HL07692 (to J.M.B.-S.). Back

2 Correspondence: Geula Gibori, Department of Physiology and Biophysics (M/C 901), University of Illinois, 835 S. Wolcott Avenue, Chicago, IL 60612-7342. FAX: 312 996 1414; ggibori{at}uic.edu Back

3 Current addresses: IUT Dijon, Bd Dr Petitjean BP 17867/UPRES Lipides et Nutrition, Universitè de Bourgogne, Facultè des Sciences de la Vie, 6 Bd Gabriel, 21000 Dijon, France Back

4 Current address: Laboratoire de Pharmacodynamie et Physiologie Pharmaceutique, Facultè de Pharmacie, BP 87900, 21079 Dijon Cedex, France Back

5 Current address: Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, 414 East Clark Street, Vermillion, SD 57069 Back

6 Current address: FDA/CFSAN, 5100 Paint Branch Pkwy, College Park, HFS-275, MD 20740 Back

Received: 25 September 2002.

First decision: 16 October 2002.

Accepted: 26 November 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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