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Endocrinology 142(9):4086 – 4094 Copyright © 2001 by The Endocrine Society
PRL Antiapoptotic Effect in the Rat Decidua Involves the PI3K/Protein Kinase B-Mediated Inhibition of Caspase-3 Activity CHRISTIAN TESSIER, ANNE PRIGENT-TESSIER, SUSAN FERGUSON-GOTTSCHALL, YAN GU, AND GEULA GIBORI Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, Illinois 60612 During gestation, the uterus undergoes severe changes to accommodate and protect the developing conceptus. In particular, stromal endometrial cells proliferate and differentiate to form the decidual tissue, which produces PRL. Once the conceptus begins to grow, extensive regression by apoptosis take place in the decidua coincident with the loss of the PRL receptor in this tissue. In this report we have established for the first time that PRL, acting through the long form of the PRL receptor and the PI3K pathway, exerts an antiapoptotic effect in rat decidua. We have also shown that protein kinase B phosphorylation on serine 473 as well as its nuclear translocation are stimulated by PRL in decidual cells. Moreover, we have found that caspase-3, a well known effector of apoptosis, becomes expressed and active in the rat decidua just at a time
A
POPTOSIS, ALSO KNOWN as programmed cell death, is an important biological process that is required to maintain the integrity and homeostasis of multicellular organisms. In the rat, implantation of the blastocyst during gestation or artificial stimuli during pseudopregnancy induces the proliferation and differentiation of endometrial stromal cells into decidual cells (1, 2). Once the blastocyst implants and begins to grow, a profound reorganization and regression of the decidual tissue take place. This regression, which is necessary for embryonic growth and for the normal progress of pregnancy, was shown to occur by apoptosis in a cell-specific and time-related manner (3–5). The antimesometrial decidual cells undergo severe regression around midpregnancy to allow for the increase in placental size and ultimately transform into a thin layer of tissue termed decidua capsularis. Cell regression later takes place in the mesometrial decidua or decidua basalis that gradually thins out as well with the progress of pregnancy. Interestingly, both decidual tissues regress with similar morphology and kinetics whether induced by the implanting blastocyst or by artificial means, suggesting that the signal for decidual cell reorganization does not emanate from the conceptus, but may involve decidual produced factor(s). We have recently shown that in the rat, as in the human, the decidua produces and secretes PRL (6) and that this tissue expresses both forms
Abbreviations: Jak, Janus tyrosine kinase; NF-B, nuclear factor-B; oPRL, ovine PRL; PKB, protein kinase B; PKB-DN, dominant-negative PKB; PRL-R, PRL receptor; PRL-RL, PRL-R long form; PRL-RS, PRL-R short form; rPL, rat placental lactogen; Stat, signal transducer and activator of transcription.
when this tissue undergoes extensive apoptosis. PRL was able to down-regulate both caspase-3 mRNA levels as well as activity. Furthermore, using a protein kinase B dominantnegative expression vector, we provide evidence that PRL inhibition of caspase-3 requires an intact protein kinase B pathway. Finally, we have also found that rat placental lactogen I and II dose-dependently inhibit caspase-3 mRNA, suggesting multiple sources of PRL in the hormonal control of rat decidual regression. In summary, the results of this study have defined an important role for decidual PRL in the normal progress of pregnancy, specifically in the regression and reorganization of the decidua. (Endocrinology 142: 4086 – 4094, 2001)
of the PRL receptors (PRL-R) (7). An antiapoptotic effect of PRL was recently shown in several cell types. In rat Nb2 lymphoma cells, PRL inhibits DNA fragmentation induced by glucocorticoids (8); and in mammary glands, GH and PRL deficiency increased DNA degradation (9). More recently, PRL was demonstrated to be a survival factor for rat dorsal and lateral prostate epithelium (10). Interestingly, our previous studies have revealed that during development extensive apoptosis occurs in the decidua at a time when the PRL-R disappears from this tissue (3, 7), and that the delayed apoptosis that occurs in the mesometrial decidua (3) is also accompanied by a delayed loss of PRL-R (7). These findings led us to investigate whether PRL plays a role in the control of apoptosis in the decidua. In this investigation we have established an antiapoptotic role for PRL in the rat decidua and have shown that this hormone can act as a survival factor, through the long form of the PRL-R and the PI3K/protein kinase B (PKB) pathway. Moreover, our results have revealed for the first time that one of the targets of PRL action in the decidua is caspase-3, whose expression and activity are down-regulated by this hormone. Materials and Methods Chemicals Acrylamide and bis-acrylamide were obtained from Accurate Chemical Inc. (Westbury, NY) and Eastman Kodak Co. (Rochester, NY), respectively; Taq DNA polymerase was purchased from Pan Vera Corp. (Madison, WI); [32P]deoxy-CTP was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL); the oligonucleotides used as primers in the RT-PCR analysis were obtained from Life Technologies, Inc. (Grand Island, NY); tissue culture medium (RPMI 1640), antibiotic-
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antimycotic solution, nonessential amino acids, and sodium pyruvate were purchased from Mediatech (Washington DC); FBS was purchased from HyClone Laboratories, Inc. (Logan, UT); trypsin-EDTA was obtained from Life Technologies, Inc.; progesterone and all other reagent grade chemicals were purchased from Sigma (St. Louis, MO); and ovine PRL (oPRL; PRL-18, 30 IU/mg) was a gift from NIDDK, NIH (Bethesda, MD). The polyclonal Ser473 phospho-PKB, total PKB, and 17-kDa active caspase-3 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). The polyclonal total caspase-3 antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY), and the secondary tetramethylrhodamine isothiocyanate-conjugated antirabbit-IgG and horseradish peroxidase-labeled antirabbit-IgG antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). AG 490, LY294002, and wortmannin were obtained from Calbiochem-Novabiochem, Corp. (San Diego, CA). Rat placental lactogen I and II (rPL-I and rPL-II) were provided by Dr. Robert Shiu (University of Manitoba, Winnipeg, Canada), and the expression vector for dominantnegative PKB (PKB-DN) was a gift from Dr. Nissim Hay (University of Illinois, Chicago, IL). The expression vectors for rat PRL receptor long and short forms were a gift from Dr. Paul Kelly (INSERM, U-344, Faculte de Medecine Necker, Paris, France).
Animal model Pseudopregnancy was induced in Holtzman female rats by mating them with vasectomized males at the Harlan facilities (Madison, WI). The day a vaginal plug was found was designated d 1 of pseudopregnancy. Rats were kept under controlled conditions of light (14 h/d; lights on, 0500 –1900 h) and temperature (22⫺24 C) with free access to standard rat chow and water. All experiments were conducted in accordance with the principles and procedures of the NIH Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care and use committee. Decidualization of uterine endometrium was induced by scratching the antimesometrial surface of both uterine horns with a hooked needle on d 5 of pseudopregnancy under ether anesthesia.
Primary cell culture Decidual cells in primary culture were obtained as previously described (11). Cells (1.2–1.5 ⫻ 106) were seeded in six-well plates and incubated at 37 C in a 95% air-5% CO2 humidified atmosphere in RPMI 1640 medium containing 2 ⫻ antibiotic-antimycotic solution (200 U/ml penicillin G, 0.5 g/ml amphotericine B, and 200 g/ml streptomycin), 1 ⫻ nonessential amino acids, 1 mm sodium pyruvate, 0.45% d-glucose, and 10% FBS. Cells were allowed to attach for 3– 4 h, washed several times with PBS, and then cultured for 12–72 h in RPMI 1640 phenol red-free medium supplemented with 1% charcoal-dextran-stripped FBS with or without treatment. When transient transfections needed to be performed, the cells were transfected using lipofectin reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. The cells were incubated with the lipofectin-reagent-DNA complexes diluted in Opti-MEM I reduced serum medium (Life Technologies, Inc.) for 4 h, after which an equal volume of RPMI 1640 phenol red-free medium containing 2% charcoal-dextran-treated FBS was added to each wells with or without treatment. Medium was changed every 12 h in the case of PRL treatment or every 24 h in the other cases. At the end of the experiment, cells were washed twice with ice-cold PBS and stored at ⫺80 C until RNA or protein extraction.
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Immunoblot analysis To isolate protein from decidual tissue, tissue was homogenized in an ice-cold lysis buffer (PBS containing 2% SDS, 2 mm EDTA, 1 mm phenylmethylsulfonylfluoride, 2 g/ml aprotinin and leupeptin, and 1 g/ml pepstatin) with a Polytron homogenizer (Brinkmann Instruments, Inc., Ontario, Canada) and then centrifuged at 10,000 ⫻ g for 10 min. An aliquot of the supernatant was kept for protein measurement. To obtain protein from cultured cells, primary cells were washed at least twice in ice-cold PBS and were scraped off the culture dishes using a rubber policeman. The cells were then resuspended in RIPA lysis buffer [50 mm Tris-Cl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonylfluoride, and 1 g/ml aprotinin, leupeptin, and pepstatin]. Protein concentrations were determined using the bicinchoninic acid kit (Pierce Chemical Co., Rockford, IL). Equal amounts of total proteins (30 – 40 g/lane) were dissolved in Laemmli buffer and heated for 5 min at 100 C. Proteins were separated through 12% SDS-PAGE gels under reducing conditions in 25 mm Tris-Cl (pH 8.3), 192 mm glycine, and 0.1% SDS in the case of PKB immunoblots or through 15% SDS-PAGE gels in 100 mm Tris-Tricine with 0.1% SDS in the case of caspase-3 blots. The latter condition allows reduction of the gel acrylamide concentration and improved migration of low mol wt proteins. Proteins were then electrophoretically transferred to nitrocellulose membranes (0.2 m pore size, Protran, Schleicher & Schuell, Inc., Keene, NH) in cold transfer buffer [20 mm Tris-Cl (pH 8.4), 192 mm glycine, and 20% methanol]. The blots were incubated for 1 h at room temperature in 5% nonfat dry milk in TBS buffer [20 mm Tris-Cl (pH 7.6) and 137 mm NaCl] containing 0.1% Tween 20 to block nonspecific binding, then washed and incubated overnight at 4 C with anti-phospho-PKB, total PKB, or active caspase-3 antibodies at a dilution of 1:1000. The membranes were washed and incubated with a horseradish peroxidase-conjugated antirabbit IgG (1: 1000) for 2 h. Protein-antibody complexes were visualized using the enhanced chemiluminescence Western blotting system (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and exposed for 1–5 min to x-ray film (Biomax MR, Kodak).
Immunocytochemistry Decidual cells in primary culture were grown for 12– 48 h in RPMI 1640 phenol red-free medium supplemented with 1% charcoal-dextrantreated FBS on sterile coverslips (13 mm in diameter) in four-well plates (Nunc, Naperville, IL). Cells were washed twice in PBS and fixed for 10 min in 4% paraformaldehyde solution in PBS at room temperature. After rinsing in TBS buffer, the cells were permeabilized for 15 min at room temperature in TBS-10% BSA, 0.1% Triton X-100, and 0.2% Tween 20 solution. The cells were then incubated overnight at 4 C with either a polyclonal antibody to total PKB or a polyclonal antibody to total caspase-3 at a 1:100 final dilution in TBS-1% BSA. Control cells were treated with TBS-1% BSA alone. The cells were then exposed for 3 h at room temperature to a tetramethylrhodamine isothiocyanate-conjugated antirabbit IgG (1:200 dilution). The coverslips were mounted in Vectashield medium (Vector Laboratories, Inc., Burlingame, CA) onto microscope slides containing a counterstain for DNA (4⬘,6-diamino-2phenylindole) and observed with a Carl Zeiss LSM 510 laser confocal microscope (Oberkochen, Germany) equipped with a ⫻40 water immersion objective lens (NA 1.2).
RNA isolation and semiquantitative RT-PCR analysis DNA ladder The internucleosomal cleavage of DNA was analyzed as follows. At the end of the experiment, cells were scratched in culture medium and centrifuged for 5 min at 1500 ⫻ g. The cell pellets were resuspended and incubated at 50 C overnight in 100 mm NaCl, 10 mm Tris-Cl (pH 8.0), 25 mm EDTA (pH 8.0), 0.5% SDS, and 0.1 mg/ml proteinase K (Life Technologies, Inc.). The digested cells were extracted for DNA with phenol/ chloroform/isoamyl alcohol (25:24:1, vol/vol/vol). The DNA was precipitated and digested for 1 h at 37 C in 1 g/ml ribonuclease solution (deoxyribonuclease-free; Roche, Indianapolis, IN). After extraction and precipitation, an equal amount of DNA (3–5 g) was separated by electrophoresis on a 1% agarose gel impregnated with ethidium bromide. The DNA pattern was examined by UV transillumination.
Total RNA from frozen decidual tissue or decidual cells in primary culture was purified using Tri-Reagent (Sigma) according to the manufacturer’s instructions. The RT and PCR reactions were performed as previously described (11). 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. Each PCR reaction included rat ribosomal protein L19 mRNA primers used as internal control to normalize the data. After autoradiography, data were quantified using a PhosphorImager and ImageQuant version 3 software (Molecular Dynamics, Inc., Sunnyvale, CA). For the detection of caspase-3 mRNA, we designed oligonucleotide primer pairs based on the sequence of the rat caspase-3 gene (5⬘-ACCGAT-
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GTCGATGCAGCTAA-3⬘ and 5⬘-GGTGCGGTAGAGTAAGCATA-3⬘) (12). L19 primers were 5⬘-CTGAAGGTCAAAGGGAATGTG-3⬘ and 5⬘CGTTCACCTTGATGAGCCCATT-3⬘ (13). The predicted sizes of the PCRamplified products were 404 and 198 bp for caspase-3 and L19, respectively.
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, cells were scratched in culture medium and centrifuged for 5 min at 1500 ⫻ g. Cell pellets were resuspended in PBS, and an aliquot was kept for protein measurement. After centrifugation, cell pellets were resuspended in ice-cold cell lysis buffer and incubated on ice for 10 min. At the end of the incubation, cell lysates were centrifuged at 10,000 ⫻ 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 OD was measured at 405 nm.
Statistical analysis Data were examined by one-way ANOVA followed by Duncan’s multiple range test. When appropriate, t test was used. P ⬍ 0.05 was accepted as statistically significant.
Results DNA fragmentation in primary decidual cells
To study the mechanism controlling decidual regression, decidual cells in primary culture obtained from d 9 pseudopregnant rats were first grown in 1% FBS-DCC for different times. Genomic DNA was isolated, and DNA fragmentation, a hallmark of apoptosis, was examined as described in Materials and Methods. The results show an increase in DNA fragmentation with time of culture (Fig. 1). Extensive DNA degradation was found after 72 h of culture, indicating that decidual cells in primary culture are an appropriate model to study the mechanisms controlling apoptosis in the decidua.
FIG. 1. DNA fragmentation in decidual cells in primary culture. Decidual cells were isolated from pseudopregnant rats (d 9 of pseudopregnancy) and cultured for different times in RPMI 1640 phenol red-free medium containing 1% charcoal-dextran-treated FBS. Genomic DNA was isolated, and equal amounts of DNA were applied on a 1% agarose gel and electrophoresed as described in Materials and Methods. The position of a 100-bp DNA ladder is shown on the right. One representative gel of three experiments is shown.
Tessier et al. • PRL Inhibits Apoptosis in the Rat Decidua
PRL inhibits apoptosis through the PI3K/PKB pathway in decidual cells in primary culture
As we have previously found that the decidual tissue undergoes extensive apoptosis just at a time when PRL-R disappears from this tissue (3, 7), we examined whether PRL had any effect on the apoptotic process that takes place in the decidua. Decidual cells in primary culture were grown for 72 h in the presence of different doses of PRL. The effect of these treatments on DNA degradation was examined. As we have previously described that decidual cells in culture lose the PRL-R within 12 h (7), we also transfected the cells with either the PRL-R long form (PRL-RL) or the PRL-R short form (PRL-RS). PRL was able to decrease DNA fragmentation significantly (40%) in cells transfected with PRL-RL at the 1 g/ml dose (Fig. 2A), but had no effect in cells transfected with PRL-RS (Fig. 2B). To determine by which signaling pathway PRL exerts its effect, we cultured the cells with or without PRL in the presence or absence of inhibitors of various signaling pathways. As PRL is known to signal through the classical Janus tyrosine kinase (Jak)/signal transducer and activator of transcription (Stat) pathway (14), we used AG490, which has been described as a specific inhibitor of Jak2 (15). However, because PRL can also activate the PI-3 kinase pathway (16) and because the PI3K was shown to play a crucial role in the prevention of apoptosis in other cell types (17), we also used two specific inhibitors of this pathway, wortmannin and
FIG. 2. Effect of PRL on DNA fragmentation in decidual cells in primary culture. Decidual cells obtained from d 9 pseudopregnant rats were transfected with 1 g/well of either PRL-RL (A) or PRL-RS (B). The cells were cultured for 72 h in presence of various doses of PRL in RPMI 1640 phenol red-free medium containing 1% charcoaldextran-treated FBS. Genomic DNA was isolated as described in Materials and Methods. The left panels show one representative ethidium bromide gel (n ⫽ 3), and the right panels depict the densitometric analysis of the DNA ladder bands (mean ⫾ SEM; values expressed as a percentage of the control, which was considered 100%). *, P ⬍ 0.05 compared with vehicle-treated controls (by one-way ANOVA, followed by Duncan’s multiple range test).
Tessier et al. • PRL Inhibits Apoptosis in the Rat Decidua
LY294002. As previously shown, a 72-h treatment with 1 g/ml PRL was able to inhibit DNA fragmentation (Fig. 3). The addition of AG490 at a concentration known to prevent PRL signaling through the Jak/Stat5 pathway in decidual cells (18) did not prevent PRL-mediated inhibition of apoptosis. In contrast, both wortmannin and LY294002, well known inhibitors of PI-3 kinase activity (19, 20), reversed the PRL effect. AG490, wortmannin, and LY294002 added alone to the culture medium had no effect on DNA fragmentation. As PKB is a known downstream target of PI3K (17), we examined whether PRL was able to activate this kinase. Decidual cells were cultured for different times in the presence of 1 g/ml PRL, and phosphorylation of PKB was examined using an antibody that specifically recognizes PKB phosphorylated on Ser473. Western blot analysis revealed that PRL was able to induce phosphorylation of PKB on Ser473 after 15 and 30 min of culture (Fig. 4A). To examine whether PRL induces PKB translocation to the nucleus, decidual cells were cultured in the presence of PRL, and PKB localization was examined by immunocytochemistry. Results shown in Fig. 4B revealed that PRL induces a redistribution of PKB from the cytoplasm to the nucleus of the decidual cells. Caspase-3 expression during pseudopregnancy
Because one of the caspases known to play a crucial role in apoptosis is caspase-3, we next examined whether this protease is expressed in decidual tissue. Figure 5A shows DNA degradation during pseudopregnancy in both antimesometrial and mesometrial decidual tissue. As previously shown (3), fragmentation of DNA was absent on d 10 in both tissues and was observed only in the antimesometrial tissue on d 12 of pseudopregnancy. By d 14, extensive DNA deg-
FIG. 3. Effects of Jak2 and PI-3 kinase inhibitors on PRL inhibition of DNA fragmentation in decidual cells in primary culture. Decidual cells in primary culture (d 9 of pseudopregnancy) transfected with 1 g/well PRL-RL were cultured in RPMI 1640 phenol red-free medium supplemented with 1% charcoal-dextran-treated FBS for 72 h in the presence or absence of oPRL (1 g/ml) with or without AG490 (20 M), wortmannin (100 nM), or LY 294002 (5 M). The effects of these different treatments on DNA fragmentation were determined as described in Materials and Methods. One representative gel of three experiments is shown.
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radation was found in both antimesometrial and mesometrial decidua, although it was more pronounced in the antimesometrial tissue. Interestingly, when we examined the expression of the 17-kDa active caspase-3, we found a correlation between the expression of this caspase and DNA fragmentation (Fig. 5B). No active caspase-3 protein could be detected on d 9 and 10 of pseudopregnancy. The expression of this protease increased thereafter, becoming highly expressed at a time when extensive apoptosis occurs in decidual tissue. When we examined caspase-3 mRNA levels in both antimesometrial and mesometrial tissue, we found low levels in the antimesometrial or no expression in the mesometrial decidua during early pseudopregnancy (d 10 –12) when no apoptosis was observed (Fig. 6). Caspase-3 mRNA expression increased from d 12 of pseudopregnancy, correlating to the abrupt increase in DNA fragmentation and active caspase-3 protein as shown in Fig. 5. Caspase-3 mRNA and activity are inhibited by PRL through the PKB pathway in decidual cells in primary culture
To determine whether PRL could affect caspase-3 expression or activity, we first examined whether decidual cells in primary culture express this protease. Decidual cells were cultured 24 h in 1% FBS-DCC. Immunocytochemistry was performed using an antibody that recognizes both the active and nonactive forms of caspase-3. As shown in Fig. 7A, caspase-3 expression was observed in decidual cells to be mainly located around the nucleus and in the filaments of the cells. Interestingly, when we examined mRNA levels of caspase-3 in decidual cells in primary culture, we observed an increase in the expression of this protease with time of culture (Fig. 7B). The maximum expression was observed after 72 h of culture, which correlates to the maximum DNA degradation seen under the same culture conditions (Fig. 1). To examine whether caspase-3 could be a target for PRL, we cultured the cells for 72 h with different doses of PRL and examined both its activity and expression. The results revealed that PRL was able to significantly decrease both the protein levels of active caspase-3 (Fig. 8A) and its activity (Fig. 8B). Interestingly, PRL was also able to significantly decrease caspase-3 mRNA levels after 12 h of culture (Fig. 8C). Because rPL-I and rPL-II, two PRL-like hormones produced by the trophoblast (21), are also known to bind to the PRL-R, we examined the roles of these two hormones in caspase-3 mRNA expression (Fig. 9). We cultured decidual cells for 12 h with different doses of rPL-I or rPL-II and examined caspase-3 expression by RT-PCR. Similarly to PRL, both rPL-I (Fig. 9A) and rPL-II (Fig. 9B) were able to decrease caspase-3 mRNA levels in a dose-dependent manner. To find out whether PRL action through the long form of the PRL-R on caspase-3 is through PKB, we transfected decidual cells with either a PKB-DN expression vector or an empty vector. Cells were then cultured for 72 h in the presence or absence of 1 g/ml PRL, and caspase-3 activity was evaluated. As previously shown, PRL significantly inhibited caspase-3 activity (Fig. 10). This PRL effect was reversed by
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Tessier et al. • PRL Inhibits Apoptosis in the Rat Decidua
FIG. 4. Effect of PRL on phosphorylation and translocation of PKB in decidual cells in primary culture. A, Decidual cells were isolated from d 9 pseudopregnant rats and cultured in RPMI 1640 phenol red-free medium supplemented with 1% charcoal-dextran-treated FBS. After 12 h of culture, the cells were treated with 1 g/ml PRL for different times. Total proteins were isolated as described in Materials and Methods, and equal amounts were separated by SDS-PAGE and transferred to nitrocellulose membrane. One representative immunoblot (n ⫽ 3) is shown. B, Decidual cells were cultured on sterile coverslips for 12 h in RPMI 1640 phenol red-free medium supplemented with 1% charcoal-dextrantreated FBS. The cells were then treated for 1 h with 1 g/ml PRL, and the coverslips were prepared for immunocytochemistry as described in Materials and Methods. a, Control cells incubated without primary antibody. Cells treated without (b) or with (c) PRL were incubated with a polyclonal PKB antibody (1:100, final dilution).
FIG. 5. DNA fragmentation and caspase-3 expression in endometrial stromal tissue during decidualization. A, Decidual tissue was obtained between d 10 and 14 of pseudopregnancy and separated into mesometrial (M) and antimesometrial (A) tissue. Genomic DNA was isolated, and equal amounts were electrophoresed as described in Materials and Methods. B, Equal amounts of total proteins (40 g) obtained from whole decidual tissue on different days of pseudopregnancy were transferred to nitrocellulose membrane and probed with a polyclonal antibody that specifically recognizes the 17-kDa active form of caspase-3. One representative immunoblot is shown. In lane C, cellular extract provided by Cell Signaling Technology, Inc., was loaded as a positive control for cleaved caspase-3.
FIG. 6. Developmental expression of caspase-3 mRNA in decidual tissue of pseudopregnant rats. Total RNA was isolated from antimesometrial (A; panel A) or mesometrial (M; panel B) decidual tissue at different stages of pseudopregnancy and analyzed by RT-PCR using specific primers for rat caspase-3 as described in Materials and Methods. Ribosomal L19 mRNA was used in each reaction as an internal standard for normalizing the data. The upper panels depict one representative autoradiogram (n ⫽ 3). The lower panels show the normalized mRNA levels as the mean ⫾ SEM
PKB-DN, indicating that PKB is involved in PRL inhibition of caspase-3. Discussion
In the present investigation we have demonstrated that PRL can act, through the long form of the PRL-R, as a survival factor able to inhibit apoptosis in the rat decidua. No PRLmediated inhibition of apoptosis was observed in decidual cells expressing only the short form of this receptor. It is well
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FIG. 7. Immunolocalization and expression of caspase-3 in decidual cells in primary culture. A, Decidual cells obtained from d 9 pseudopregnant rats were cultured for 48 h in RPMI 1640 phenol red-free medium supplemented with 1% charcoal-dextran-treated FBS. Immunocytochemistry was performed as described in Materials and Methods using a polyclonal antibody that recognizes total (active and nonactive) caspase-3 (1:100, final dilution). A negative control obtained by omitting the primary antibody incubation step from the procedure was free of staining (a). A positive signal was indicated by the red staining (b). B, Total RNA obtained from decidual cells cultured for different times in RPMI 1640 phenol red-free medium supplemented with 1% charcoal-dextran-treated FBS was prepared and subjected to semiquantitative RT-PCR analysis. PCR products were visualized by autoradiography and normalized to the amount of the L19 mRNA internal control. The upper panel shows a representative autoradiogram (n ⫽ 3), and the lower panel illustrates the normalized mRNA levels (mean ⫾ SEM).
established that after PRL stimulation, PRL-R is tyrosyl phosphorylated and activates Jak2 (14). This kinase phosphorylates the cytokine Stats such as Stat1, -3, and -5 (a and b) that regulate the expression of various genes (reviewed in Ref. 22). Moreover, PRL has also been shown to activate other kinases such as MAPK (14) and PI3K (16). In rat decidual cells, PRL appears to exert its survival effect through activation of the PI3K signaling pathway and does not require Jak2 stimulation. Indeed, the specific PI-3 kinase inhibitors, wortmannin and LY294002, prevented PRL inhibition of DNA fragmentation, whereas AG490, a Jak2 inhibitor, had no effect. Although the PRL-R was shown to be phosphorylated by Jak2 in COS cells, thereby providing docking sites for the p85 PI3K subunit and activating PI3K and its downstream biological effects (23), activation of the PI3K pathway was shown to be independent of Jak2 in Nb2 cells (24), where Cbl was suggested to function as an adapter protein (25). In the rat decidua the mechanism of action by which PRL activates the PI3K pathway remains to be investigated. One of the important downstream targets of PI3K is PKB, or Akt, which has been identified as an important component of prosurvival signaling pathways (26 –28). This serine/threonine kinase is activated via the PI-3 kinase signaling pathway by a number of receptors, such as insulin, epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, or the cytokines IL-2, IL-3, and IL-4 (26 –28). In this report we provide evidence that PRL activates PKB and induces both phosphorylation and nuclear trans-
location of this kinase in decidual cells. Interestingly, GH, which, like PRL, signals through the same cytokine superfamily of receptors and activates Jak2 in response to ligand binding (29), was also shown to inhibit apoptosis through an activation of the PI-3 kinase/PKB pathway. However, whereas the GH survival effect is also dependent on Jak2 activation (30), our results suggest that PRL action may be independent of this pathway, although this hormone activates and phosphorylates Jak2 in decidual cells (31). Both human and rat decidua express the PRL gene, and it is highly possible that decidual PRL prevents cell death in both species. However, the mechanism through which this hormone exerts its protective effect is still not known. PRL was shown to up-regulate both the mRNA and protein levels of the protooncogene bcl-2, which is known to suppress cell death, and to reduce the expression of proapoptotic Bax protein in Nb2 cells (32). However, addition of PRL to glucocorticoid treatment failed to maintain normal levels of Bcl2 (33). PRL may block glucocorticoid-induced cell death by inhibiting the disruption of the mitochondrial membrane (33). In addition to Bcl2 family members, the caspase family of proteases is known to play a central role in apoptosis (34 –37). Caspases can be subdivided into apoptotic initiators and apoptotic executioners. The former caspases cleave and activate the latter, which, in turn, are responsible for degradation of essential cellular components. Among the executioner proteases, caspase-3 plays a direct role in proteolytic digestion of cellular proteins responsible for progression to
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FIG. 8. Effect of PRL on caspase-3 expression and activity in cultured primary decidual cells. A, Decidual cells transfected with the PRL-RL were cultured for 72 h in the presence of different doses of PRL in RPMI 1640 phenol red-free medium supplemented with 1% charcoaldextran-treated FBS. Total proteins were separated, and Western blotting analysis was performed using a polyclonal antibody that recognizes specifically the 17-kDa active form of caspase-3 as described in Materials and Methods. In lane C, cellular extract provided by Cell Signaling Technology, Inc., was loaded as a positive control for cleaved caspase-3. B, Caspase-3 activity was measured using the CLONTECH Laboratories, Inc., kit according to the manufacturer’s instructions from decidual cells cultured for 72 h in RPMI 1640 phenol red-free medium supplemented with 1% charcoal-dextran-treated FBS and treated with different doses of PRL. *, P ⬍ 0.05, by one-way ANOVA, followed by Duncan’s multiple range test. C, Total RNA obtained from decidual cells in primary culture (d 9 of pseudopregnancy) treated 12 h with different doses of oPRL was isolated, reverse transcribed into single stranded complementary DNA, and amplified with specific oligonucleotide pairs for caspase-3 mRNA as described in Materials and Methods. One representative autoradiogram is shown in the left panel. The densitometric analysis from three independent experiments (mean ⫾ SEM; values expressed as a percentage of the control, which was considered 100%) is depicted in the right panel. *, P ⬍ 0.05, by one-way ANOVA, followed by Duncan’s multiple range test.
apoptosis. Caspase-3 is synthesized as an inactive 32-kDa proenzyme that is activated after cleavage at specific aspartate sites (35, 36). Our study demonstrates for the first time that one of the targets of PRL action is caspase-3. Indeed, PRL was able to down-regulate both caspase-3 expression and activity. Interestingly, our results also show that caspase-3 expression in the rat decidua in vivo correlates to the disappearance of the PRL receptor from this tissue and with decidual regression by apoptosis (3, 7). This result further supports the possibility that PRL-mediated inhibition of apoptosis may at least in part occur through the suppression of caspase-3 activity. Of interest was our finding that PRLmediated inhibition of caspase-3 involves PKB. Similar to PRL, the survival action of epidermal growth factor is associated with a PI3K-dependent inhibition of caspase-3 activity in an epithelial tumor cell line (38). The serine/threonine kinase PKB mediates cell survival in a variety of systems by acting on cellular proteins with well established roles in apoptosis (27–28). In particular, PKB-induced phosphoryla-
Tessier et al. • PRL Inhibits Apoptosis in the Rat Decidua
FIG. 9. Effects of rPL-I and rPL-II on caspase-3 expression. Decidual cells were isolated from pseudopregnant rats (d 9 of pseudopregnancy) and cultured for 12 h in the presence of different doses of rPL-I or rPL-II in RPMI 1640 phenol red-free medium supplemented with 1% charcoal-dextran-treated FBS. Total RNA was prepared and subjected to RT-PCR analysis as described in Materials and Methods. RT-PCR products were visualized by autoradiography and normalized to the amount of the L19 mRNA internal control. A, The effect of rPL-I on caspase-3 mRNA; B, the effect of rPL-II. The upper panels depict one representative autoradiogram (n ⱖ 3), and the right panels show the normalized mRNA levels (mean ⫾ SEM). *, P ⬍ 0.05 compared with vehicle-treated controls (by one-way ANOVA, followed by Duncan’s multiple range test).
FIG. 10. Prevention of PRL-mediated inhibition of caspase-3 activity in decidual cells by PKB-DN. Decidual cells isolated from d 9 pseudopregnant rats were transiently transfected with PRL-RL (1 g/ well) and either pcDNA (EV; 5 g/well) or an expression vector for PKB-DN (5 g/well). The cells were then cultured for 72 h with or without 1 g/ml oPRL and caspase-3 activity was measured as described in Materials and Methods. *, P ⬍ 0.05, by one-way ANOVA, followed by Duncan’s multiple range test.
tion of caspase-9 has been found to decrease apoptosis by directly inhibiting protease activity (39). Our investigation suggests that PKB may also exert its antiapoptotic effects through an inhibition of caspase-3 activity. Interestingly, PKB was recently shown to activate NF-B in various cell types (40 – 42). NF-B is known as an antiapoptotic transcription factor, in particular by activating the inhibitor of apoptosis proteins (43, 44) that can directly inhibit caspase-3 (45). Whether PKB acts directly, as it does on caspase-9, or indi-
Tessier et al. • PRL Inhibits Apoptosis in the Rat Decidua
rectly through NF-B to inhibit caspase-3 in the rat decidua remains to be investigated. In addition to PRL, progesterone appears to protect decidual cells from apoptosis (5, 46 – 48). Two recent investigations using the PRL-R knockout mice have shown that progesterone administration to PRL-R⫺/⫺ mice rescues both implantation and decidualization failures (49, 50). However, the maintenance of full-term pregnancy was incomplete, and abortion occurred, leading the researchers (50) to suggest a direct role of PRL on the decidua. Whether the embryo resorbtions observed in the PRL-R⫺/⫺ mice are due to increased and disorganized apoptosis in the decidua is a subject of further investigation. In summary, the results of our investigation have revealed an important antiapoptotic role for PRL in the decidua. As the rat decidua expresses the PRL gene and produces PRL (6), our study provides a possible new local role for decidual PRL as an antiapoptotic molecule. The inverse correlation between the occurrence of apoptosis and the loss of PRL-R in the mesometrial and antimesometrial decidua (3, 7) strongly suggests that it is the disappearance of the PRL-R first from the antimesometrial then from the mesometrial decidua that allows for cell death at different times and at different intensities in this tissue, leading to reorganization of the decidua and allowing for the normal growth of the conceptus. Acknowledgments We are grateful to Dr. Robert Shiu for providing the rPL-I and -II, and to Dr. Nissim Hay for the dominant-negative expression vector of PKB. We thank the NIDDK and the National Hormone and Pituitary Program, NIH, for the oPRL. We also thank Dr. Catherine Boyer for skillful assistance with the confocal studies. We are very grateful to Gil B. Gibori for his help with the editing and preparation of the manuscript. Received February 16, 2001. Accepted May 15, 2001. Address all correspondence and requests for reprints to: Dr. Geula Gibori, Department of Physiology and Biophysics (M/C 901), University of Illinois, 835 South Wolcott Avenue, Chicago, Illinois 60612-7342. Email:
[email protected]. This work was supported by NIH Grants HD-12356 and HD-11119 (to G.G.) and the Ernst Schering Research Foundation (to C.T.).
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Erratum In the article by Hrabovszky, E., et al., “Estrogen receptor- immunoreactivity in luteinizing hormonereleasing hormone neurons of the rat brain” (Endocrinology 142:3261–3264, 2001), Fig. 2 appeared incorrectly because of a white dividing line inserted in panels E3 and E4. The correct figure and its legend appear on the next page. The printer regrets the error.
