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research-article |
Departments of Biochemistry and Molecular Biology,3 Obstetrics and Gynecology,4 and Medicine,5 College of Medicine, Hanyang University, Seoul 133-791, Korea
The Research Center for Bio-Medical Resources,6 Paichai University, Taejon 302-735, Korea
ABSTRACT
Using primary cell cultures of human endometrial stromal cells (ES cells), we investigated the role of phospholipase D (PLD) in 8-Br-cAMP-induced decidualization, which involves morphological and biological differentiation processes. When treated with 0.5 mM 8-Br-cAMP for 12 days, ES cells were transformed into a decidualized morphology and produced significant amounts of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1). Simultaneously, the activity and expression levels of PLD1 increased. In addition, removal of 8-Br-cAMP from decidualized ES cells restored the undifferentiated state, and this was accompanied by decreases in PLD1 promoter activity and PLD1 expression. Overexpression of dominant negative (DN)-PLD1 inhibited the morphological changes induced by 0.5 mM 8-Br-cAMP, whereas PLD1 overexpression induced morphological changes in the absence of 0.5 mM 8-Br-cAMP treatment. Moreover, knockdown of PLD1 by siRNA and blockage of PLD by treatment with 0.3% 1-butanol decreased PRL/IGFBP1 mRNA expression, whereas PLD1 overexpression increased PRL/IGFBP1 mRNA expression. Treatment of ES cells with phosphatidic acid (PA) for 3 days induced PRL mRNA expression and morphological changes, which implies that PA is an end-product of PLD activation-induced decidualization. In addition, pretreatment of ES cells with mepacrine decreased PRL/IGFBP1 expression and inhibited morphological change, whereas pretreatment with propranolol caused no changes, as compared to cAMP-treated cells, which suggests that PA induces decidualization through phospholipase A2 (PLA2G1B). Taken together, these results suggest that PLD1 regulates 8-Br-cAMP-induced decidualization through PLA2G1B, and that PLD1 upregulation is essential for the decidualization of ES cells.
8-Br-cAMP, developmental biology, decidualization, human endometrial stromal cell, phospholipase
A satisfactory reproductive outcome requires synchronous growth and maturation of the endometrium throughout the menstrual cycle. The decidualization of human endometrial stromal cells (ES cells), which develop in the late secretory stage of the menstrual cycle, is characterized by morphological and functional differentiation. ES cells around blood vessels begin to differentiate into a larger and rounder decidual phenotype on the 23rd day in women with a regular 28-day menstrual cycle [1]. Following implantation, decidualization persists and extends throughout the endometrium, leading to the formation of pregnancy deciduas. This morphological change is accompanied by the expression of several factors, such as prolactin (PRL) and insulin-like growth factor-binding protein 1(IGFBP1) [2–5]. These factors are correlated with decidualization and in turn, act as regulators of both decidualization and trophoblast functions. Thus, decidualization is necessary for normal embryo implantation and the maintenance of pregnancy [6].
There is abundant clinical and experimental evidence in support of a critical role for progesterone in maintaining the decidual phenotype. However, recent reports have suggested that the expression of decidua-specific genes may not be transcriptionally regulated by the activated progesterone receptor (PR) [7]. Furthermore, progesterone is a very weak inducer of the decidual phenotype in primary ES cells [1], and initiation of the decidual process requires both a high level of intracellular cAMP and sustained activation of the protein kinase A (PKA) pathway [8, 9]. Therefore, our present knowledge regarding the intracellular signaling pathways for decidualization is preliminary at best.
Over the past two decades, it has been extensively documented that phospholipase D (PLD) plays an essential role in the signal transduction of mammalian cells [10]. PLD catalyzes the hydrolysis of phospholipids at the terminal phosphodiester bond, thereby producing phosphatidic acid (PA) and releasing the free polar head group. PA can also be metabolically converted to diacylglycerol (DAG) by phosphatic acid phosphatase type 2A (PPAP2A), and to lyso-PA or arachidonic acid by phospholipase A2 (PLA2G1B). Both of these factors serve as second messengers that contribute to the effects of PLD. Recently, two PLD isozymes (PLD1 and PLD2) have been cloned and characterized from mammalian cells [11, 12]. PLD1 is activated by protein kinase C (PKC) and small GTP-binding proteins, such as ADP-ribosylation factor (ARF)-, and Ral- and Rho-family GTPases. In contrast, PLD2 activity is known to be constitutively active in vitro and its activity is elevated by fatty acid, although it is not affected by GTPases or PKC-
. Both PLD isoforms require phosphatidylinositol-4,5-bisphosphate (PIP2) as a cofactor for their activities [11, 12]. PLD appears to play a significant role in a broad range of physiological processes, including both short-term (e.g., secretion and superoxide generation) as well as long-term (e.g., cell proliferation, differentiation, and apoptosis) responses. Recently, a role for PLD in cell differentiation has been proposed; Marjou et al. have reported that the activity and expression of PLD increase with maturation and differentiation in human myeloid cells [13], up-regulation of PLD1 [14] and PLD2 [15] mRNA has been reported in differentiated HL60 cells, and regulated transcription of PLD1 and/or PLD2 has been observed in C6 glioma cells [16] and differentiating keratinocytes [17]. Taken together, these reports suggest a functional role for PLD in differentiation. Despite a large body of data that suggests that PLD is a pivotal factor in differentiation, it is not known whether PLD has a role in the differentiation of the endometrium.
In the present study, we investigated the role of PLD in 8-Br-cAMP-induced decidualization of human endometrial stromal cells in vitro. To the best of our knowledge, this is the first evidence that PLD1 is a regulator for decidualization, and that PLD1 activation may contribute to embryo implantation and maintenance of pregnancy in human endometrial stromal cells.
Materials for tissue culture, such as fetal bovine serum (FBS), penicillin, streptomycin, and Dulbecco modified Eagle medium (DMEM, low glucose), were obtained from Gibco-BRL (Gaithersburg, MD), and 8-Br-cAMP was from Sigma (St. Louis, MO). [3H]-palmitic acid was obtained from DuPont-New England Nuclear (Boston, MA). The following antibodies were as follows: anti-ACTB (Cell Signaling), anti-GFP monoclonal antibody (Roche, Indianapolis, IN), and a polyclonal antibody that recognizes both PLD1 and PLD2 (provided by Dr. D.S. Min, Pusan National University, Korea). L--PBt was obtained from Avanti Polar Lipid Inc. (Alabaster, AL), and the silica gel 60A plates for TLC were purchased from Whatman (Clifton, NJ). All other chemical reagents were of analytical grade.
Isolation and Culture of ES Cells
Human endometria were obtained by hysterectomy from 20 normally cycling pre-menopausal women, aged 35 to 44 years, who underwent surgery for non-endometrial abnormalities at Hanyang University Hospital between January 2004 and June 2006. Samples were collected under protocols approved by the Institutional Review Board of Hanyang University Hospital. Written informed consent was obtained from all participants. A portion of each endometrial specimen obtained was examined histologically. ES cells were isolated as described previously [18]. Briefly, tissue samples were collected in DMEM that contained 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 10% (vol/vol) fetal calf serum. After cleaning and trimming to remove blood clots and mucus, specimens were minced to fragments of less than 1 mm in size under a laminar flow hood, and then digested at 37°C for 60 min with 0.25% (wt/vol) collagenase I (Sigma) and DNase (Sigma). The cell suspension was filtered twice through a 40-µm sieve (BD Falcon). After enzymatic digestion, most of the ES cells were present as single cells or small aggregates, and they were purified. The purity of the ES cells obtained by this method was usually >90%, as determined by immunocytochemical staining of vimentin (stromal cell marker). The purified ES cells were washed, and the number of viable cells was counted by trypan blue exclusion. The viability of the isolated cells was at least 90% in each experiment. The ES cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air with 10 ml DMEM that was supplemented with 10% (vol/vol) FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin.
In Vitro Decidualization of Human ES Cells
ES cells were cultured to confluence in 100-mm culture dishes. To induce decidualization in vitro, the cells were then cultured with 0.5 mM 8-Br cAMP, which is a decidualization inducer, for 3–12 days. The culture medium was changed every three days with continuous supplementation with 8-Br-cAMP. Phase contrast microscopy was used to verify morphological changes associated with differentiation in response to 8-Br-cAMP.
PLD activity was determined as previously described by measuring [3H]-PBt produced in [3H]-palmitic acid-labeled cells via PLD-catalyzed transphosphatidylation [19]. Briefly, ES cells cultured in 6-well plates were metabolically labeled with 1 µCi/ml of [3H]-palmitate for 24 h. The cells were then pretreated with 0.3% (vol/vol) 1-butanol for 15 min, and were quickly washed with ice-cold PBS and suspended in ice-cold methanol. Lipids were extracted according to the method of Bligh and Dyer [20], and [3H]-PBt was separated from other phospholipids by thin layer chromatography on Silica G-60 plates, using a solvent system of ethyl acetate/iso-octane/acetic acid/water (26:4:6:20, v/v). The regions that corresponded to the authentic PBt bands were identified using 0.002% (wt/vol) primulin in 80% (vol/vol) acetone, scraped, and then counted using a scintillation counter.
ES cells were scraped into ice-cold phosphate-buffered saline (PBS) and harvested by microcentrifugation. The cells were then resuspended in buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1% [vol/vol] Triton X-100, 1 mM PMSF, 1 mM Na3VO4) and disrupted by sonication. Proteins were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h with TTBS (Tris-buffered saline with 0.01% [vol/vol] Tween 20) that contained 5% (wt/vol) non-fat dry skim milk, and then incubated for an additional 1 h with the primary antibodies (1 µg/ml). Unbound primary antibodies were removed by three washes (10 min each) with TTBS. The blots were then incubated with horse radish peroxidase (HRP)-conjugated secondary antibody (1:2000; New England Biolabs, Beverly, MA), and specific bands were detected by ECL (Amersham Pharmacia Biotech).
For immunocytochemical tests, ES cells were plated on a glass coverslip that was coated with fibronectin. Cells were fixed with 4% (wt/vol) paraformaldehyde/0.15% (wt/vol) picric acid in PBS, and then incubated overnight with primary antibody at 4°C. The anti-green fluorescent protein (GFP) monoclonal antibody was used at 1:400 (Roche Molecular Biochemical, Basel, Switzerland). For detection of the primary antibody, fluorescently labeled (Cy3) secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) were used according to the specifications of the manufacturer. Cells were mounted in Vectashield with DAPI (mounting medium for fluorescence analysis; Vector Laboratories, Burlingame, CA) and photographed using a fluorescent microscope (Nikon).
Construction of the Expression Vector
Lipase-inactive mutant rPLD1 cDNA was provided by J.H. Exton (Vanderbilt University, Nashville, TN). Briefly, the N-terminal Xpress-tagged rPLD1 was created by PCR amplification of the coding region that corresponds to amino acid residues 1 to 1036, and subcloned into the KpnI/XbaI sites of the polylinker region in the pcDNA3.1 vector. rPLD1-V5 was also generated by PCR amplification, followed by subcloning into the HindIII/XbaI site within the same frame as the C-terminal V5 tag of the pcDNA3.1/V5-HisA vector. The HindIII/KpnI fragment of the C-terminal-tagged rPLD1 was subcloned into the polylinker region of the pEGFP-C1 vector, to generate the N-terminal EGFP-tagged rPLD1-V5. ES cells grown for 4–6 days were transfected using the Nucleofector Kit (Amaxa), according to the manufacturers protocol. Transfection controls were carried out with the same amount of vector. Twenty-four hours after transfection, the cells were differentiated by treatment with 0.5 mM 8-Br-cAMP for the indicated day. Western blot analysis for the expression of plasmid DNA was used as an internal control, to determine the transfection efficiency.
Small Interfering RNA Sequences
The single siRNA for PLD1 was purchased from Dharmacon Research Inc. (Lafayette, CO) and the second siRNA for human PLD1 was a kind gift from Sung Ho Ryu (Pohang University of Science and Technology, Korea) [21]. A BLAST search of all siRNA sequences revealed no significant homology to any other sequences in the database. Then, 200 nM of the siRNA duplex was transiently transfected into human ES cells using the Nucleofector kit. Transfection controls were carried out with the same amount of EGFP-expressing vector. Twenty-four hours after transfection, the cells were differentiated by treatment with 0.5 mM 8-Br-cAMP for an additional 2 days in vitro.
The PLD1 reporter plasmid was constructed with the human PLD1 promoter fragment cloned upstream of the luciferase cDNA in the pGL3-Basic vector (Promega). ES cells were transfected transiently with pGL3-Basic, the promoterless vector or the PLD1 reporter plasmid (pGL3-PLD1p-1865/+9) using the Nucleofector Kit. To correct for the variance of transfection efficiency in each assay, the pCMV-ß-gal vector was cointroduced. One day after transfection, the cells were treated with 0.5 mM 8-Br-cAMP for 2 days, to induce decidualization. Gene expression was analyzed using a firefly luciferase assay system (BD-Pharmingen) and a microplate luminometer (Berthold). The relative firefly luciferase activity was defined as the luciferase activity normalized for ß-galactosidase activity in each corresponding cell lysate.
RNA Extraction and Reverse Transcription-PCR (RT-PCR) Analysis
Total cellular RNA was prepared using Tri Reagent (Molecular Reagent Center, Cincinnati, OH), according to the recommendations of the manufacturer. The Superscript Kit (Invitrogen) was used for cDNA synthesis. PCR reactions were carried out according to standard protocols. Initially, the optimal MgCl2 concentrations and cycle numbers for a linear amplification range were determined. The primer sequences (forward and reverse) were as follows: ACTB (5'-CCCAGGCACCAGGGCGTGATC-3' and 5'-TCAAACATGATCTGGGTCAT-3'); PRL (5'-GCCCCCTTGCCCATCTGTCC-3' and 5'-AGAAGCCGTTTGGTTTGCTCC-3'); IGFBP1 (5'-TGCTGCAGAGGCAGGGAGCCC-3' and 5'-AGGGATCCTCTTCCCATTCCA-3'); hPLD1 (5'-ACTCTGTCCAAAGTTAACATGTCACTG-3' and 5'-GGCTTTGTACTTGAGCAGCTCTCT-3'). The RT-PCR products were analyzed using an ethidium bromide-containing agarose gel.
All experiments were performed at least three times, and the data were analyzed using one-way ANOVA with statistical significance set at the level of P < 0.05.
Culture and Decidualization of Human Endometrial Stromal Cells
ES cells were separated from epithelial cells by digesting the tissue fragments with collagenase, as described above. The cells, which were spindle-shaped when plated, reached confluence after 3–6 days of culture, and retained a fibroblast-like appearance. Decidualization was then induced by incubating subconfluent cells in medium that contained 0.5 mM 8-Br-cAMP and replacing the supernatant every 3 days with fresh culture medium containing 0.5 mM 8-Br-cAMP. After growing in the presence of 0.5 mM 8-Br-cAMP for 6 days, the cells were transformed into large polygonal cells with enlarged nuclei and increased amounts of cytoplasm, resembling decidual cells in vivo (Fig. 1A). As the cells proceeded to differentiation, their borders became less distinct because of extracellular matrix formation. With further differentiation, the cells began to form aggregates and increased in size and number. These morphological features were maintained throughout the culture periods. To confirm in vitro decidualization, the levels of PRL and IGFBP1 expression were determined by RT-PCR, since these genes show increased expression during decidualization [22]. As shown in Figure 1B, treatment with 8-Br-cAMP increased PRL and IGFBP1 mRNA expression within 3 days, and the increased expression was maintained throughout the culture period. Therefore, these results suggest that PRL and IGFBP1 can be used as decidualization markers.
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Changes in PLD Activity and Expression Levels during Decidualization
To examine the role of PLD in decidualization, we first measured PLD activity after treatment of ES cells with 8-Br-cAMP to induce decidualization. As 8-Br-cAMP-induced decidualization progressed, PLD activity was increased by up to 550% (Fig. 2A). However, in the absence of 8-Br-cAMP, the morphology and PLD activity of the ES cells did not change during the same time period (data not shown). To confirm PLD activation during in vivo decidualization, we investigated the levels of PLD activity in ES cells isolated from a 5-mo-pregnant uterus, and found that the basal PLD activity in these ES cells was increased to 200% compared to ES cells isolated from a nonpregnant uterus (Fig. 2B). These results demonstrate that PLD activity is upregulated during in vivo and in vitro decidualization.
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Next, we investigated whether this increase in PLD activity was due to increased biosynthesis of PLD protein. As shown in Figure 3A, the level of PLD1 protein increased gradually following 8-Br-cAMP treatment, peaking after 9 days (up to 260% higher than the control cells), whereas PLD1 expression in the absence of 8-Br-cAMP did not change during the same time period (data not shown). In contrast, the level of PLD2 protein remained relatively constant during 8-Br-cAMP-induced decidualization. To confirm further the increase in PLD1 expression, we examined the effect of decidualization on PLD1 promoter activity. Thus, we transfected ES cells with the luciferase reporter gene under the control of the full-length PLD1 promoter and pCMV-ß-gal gene. After 24 h, the cells were treated with 8-Br-cAMP for an additional 2 days, and luciferase was assayed. Figure 3B shows that treatment of the cells with 8-Br-cAMP markedly enhanced PLD1 promoter activity by up to 8.4 fold, which demonstrates that the increased level of PLD1 was due to the activation of the PLD1 promoter. Therefore, the observed correlation between decidualization and PLD1 upregulation in ES cells indicates that PLD1 is involved in the decidualization of ES cells.
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Effects of 8-Br-cAMP Withdrawal on PLD1 Expression and Morphology
Upon withdrawal of 8-Br-cAMP stimulation, decidualized ES cells are restored to an undifferentiated phenotype and cease to express differentiation markers, such as PRL and IGFBP1 [5]. Therefore, to verify that the upregulation of PLD1 was a downstream event of 8-Br-cAMP stimulation, we withdrew 8-Br-cAMP from the cultures of fully decidualized ES cells after treatment with 8-Br-cAMP for 9 days. Microscopic examination revealed that an undifferentiated morphology appeared 5 days after 8-Br-cAMP withdrawal (Fig. 4A). In addition, RT-PCR analysis showed that the mRNA expression levels of PRL and IGFBP1 decreased according to the timing of 8-Br-cAMP withdrawal (Fig. 4B). Western blot analysis revealed that PLD1 protein expression decreased concomitant with withdrawal of 8-Br-cAMP for 6 days (Fig. 4C). We also investigated whether PLD1 protein expression changed in response to 8-Br-cAMP withdrawal. As shown in Figure 4D, when ES cells were treated with 8-Br-cAMP for 2 days, PLD1 promoter activity was increased up to 8-fold compared to the control cells. However, after treatment with 8-Br-cAMP for 1 day and 8-Br-cAMP withdrawal for 1 day, PLD1 promoter activity decreased by 70%, compared to cells that were treated with 8-Br-cAMP for 2 days. This result suggests the coupling of PLD1 expression with decidualization. Taken together, these results confirm that the level of PLD1 is related to decidualization, and that PLD1 is a decidualization marker in ES cells.
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Effects of DN-PLD1 and WT-PLD1 Overexpression on Decidualization
In order to confirm the effect of PLD activity on decidualization, we transfected ES cells with the dominant-negative (DN)-PLD1 or PLD1 expression plasmid. Thus, DN-PLD1 and PLD1 DNA was inserted into the EGFP vector, and the recombinant plasmid was electroporated into ES cells. As shown in Figure 5A, ES cells were successfully transfected with DN-PLD1 or PLD1 by electroporation. Furthermore, the overexpression of exogenous PLD1 increased the basal PLD activity 1 day after transfection, whereas the overexpression of DN-PLD1 decreased PLD activity (Fig. 5B). During 8-Br-cAMP-induced decidualization, the PLD activity of the PLD1-overexpressing cells was significantly higher than that of the EGFP-transfected cells. However, the PLD activity of the DN-PLD1-transfected cells decreased to the basal level.
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Next, we examined the transfected cells under a fluorescence microscope, to examine the effect of gene transfection on cell morphology. The transfected cells were easily visible under the fluorescence microscope owing to their green color. One day after transfection, the control and DN-PLD1 transfectants showed very few cells with morphological changes, whereas 23% of the PLD1-transfected cells exhibited altered morphology (Fig. 6, A and B), which suggests that PLD activity induces morphological changes. Subsequently, the transfected cells were cultured with or without 0.5 mM 8-Br-cAMP. As shown in Figure 6B, the percentage of cells with the decidualized morphology significantly increased by 72% after treatment of the control cells with 8-Br-cAMP for 9 days, whereas only 23.8% of the cells changed in the absence of 8-Br-cAMP treatment. On the other hand, the DN-PLD1 transfectants showed very few morphological changes in the presence of 8-Br-cAMP for 9 days, and only 20% of the cells were transformed into the decidualized morphology. Accordingly, these results indicate that morphological change is inhibited by the blockage of PLD1 activation. When we cultured the PLD1-transfected cells in the presence of 8-Br-cAMP, they were rapidly transformed into the decidualized morphology (Fig. 6B). Treatment with 8-Br-cAMP for 3 days changed 72% of the PLD1-transfected cells into the decidualized morphology, whereas only 31% of the control cells were altered. This morphological change was markedly potentiated by PLD1 overexpression, which increased up to 87% with 8-Br-cAMP treatment for 9 days (Fig. 6B). Moreover, 73% of the PLD1-transfected cells were transformed into large polygonal cells in the absence of 8-Br-cAMP for 9 days. In order to ascertain further the effect of PLD activity on decidualization, we determined the expression levels of PRL and IGFBP1, which are known decidualization markers. One day after transfection of the cells with siRNA directed against PLD1 or with PLD1, the cells were treated with 8-Br-cAMP for 2 days, and the PRL and IGFBP1 mRNA levels were then examined by RT-PCR. As shown in Figure 7, A and B, transfection with PLD1 siRNA decreased PRL and IGFBP1 expression, i.e., transfection with PLD1 siRNA(1) decreased PRL expression by 47% and IGFBP1 expression by 44%, and transfection with PLD1 siRNA(2) decreased PRL by 46% and IGFBP1 expression by 44%. However, transfection with PLD1 upregulated PRL expression by 212% and IGFBP1 expression by 210% (Fig. 7, C and D). Moreover, pretreatment for 18 h with 0.3% 1-butanol, which is a PLD inhibitor, completely abolished PRL and IGFBP1 expression, whereas pretreatment with 0.3% 2-butanol for 18 h did not induce any change (Fig. 7E). These results suggest that PLD1 upregulation is important for 8-Br-cAMP-induced decidualization.
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Effects of Treatment with PA and Pretreatment with Mepacrine or Propranolol on Decidualization
To confirm the role of PLD1 in decidualization, we treated the cells with phosphatidic acid (PA), which is one of the enzymatic products of PLD. When treated with the indicated concentration of PA for 3 days, both PRL expression (Fig. 8A) and morphological changes (Fig. 8B) were induced, which implies that PA produced by PLD activation induces decidualization. PLD-induced PA is usually degraded to DAG by phosphatidic acid phosphatase (PPAP2A) or to LPA/AA by PLA2G1B [10]. In order to determine how PA regulates decidualization, we pretreated the cells with propranolol, PPAP2A inhibitor, or mepacrine, PLA2G1B inhibitor for 1 h. As shown in Figure 8, C and D, pretreatment with propranolol for 1 h had no effect on PRL and IGFBP1 expression or morphological change. However, pretreatment with mepacrine for 1 h decreased PRL and IGFBP1 expression as well as morphological change, which suggests that PLD1 induces decidualization through PLA2G1B.
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Decidualization is a process during which endometrial cells undergo dramatic changes in their biochemical properties and morphological appearance. However, intracellular signaling pathways leading to decidualization are still far from clear. In the present study, we tried to determine the contribution of PLD to 8-Br-cAMP-induced decidualization.
The significance of PLD activity in cell maturation and differentiation has been suggested, although the role of PLD in the differentiation processes remains speculative. In the present study, the levels of PLD activity as well as the expression levels of PLD1 increased in ES cells as 8-Br-cAMP-induced decidualization progressed. Moreover, the PLD activity of ES cells from 5-mo-pregnant deciduas was upregulated about 2-fold compared to that in ES cells from nonpregnant deciduas, which indicates that PLD activity is upregulated during in vivo decidualization. Recently, Brar et al. [23] observed by microarray analysis that PLD1 was induced 3.6-fold during decidualization, which supports our finding that PLD1 mRNA was increased under decidualization conditions. Based on the following experimental data, we propose that PLD activation plays a pivotal role in decidualization. First, withdrawal of 8-Br-cAMP from the culture medium after 9 days decreased PLD1 promoter activity and PLD1 protein expression, and this was accompanied by a morphological change whereby the original cell morphology was restored. Second, DN-PLD1 transfection into ES cells almost completely inhibited the appearance of the decidualized morphology induced by 8-Br-cAMP. However, overexpression of PLD1 induced rapid change in the cells to the decidualized morphology in the absence of 8-Br-cAMP. Third, PLD1 blockage by PLD1 siRNA and 0.3% 1-butanol decreased the PRL and IGFBP1 mRNA expression levels, whereas PLD1 overexpression increased these levels. Fourth, PA, which is an enzymatic product by PLD, induced morphological change and PRL expression. Taken together, our results strongly support the notion that PLD1 activation and increased PLD1 expression are correlated with decidualization.
Multiple lines of evidence suggest that a PKA signal transduction pathway mediates the action of cAMP to induce decidualization [8, 9, 24–26]. The cAMP signal to the nucleus is transduced almost exclusively by the binding of cAMP to the R subunit of PKA and subsequent phosphorylation of nuclear target proteins by released and translocated C subunits [27]. During decidualization, elevated levels of cellular cAMP lead to elevated kinase activity and decreased R/C subunit ratio for PKA. In addition, the endometrial PKA system does not desensitize to the chronic stimulus. Therefore, the cAMP/PKA pathway may contribute to decidualization-induced gene expression. Brar et al. [23] have used by microarrays to identify 281 genes that are dynamically regulated in decidual ES cells during in vitro decidualization that is induced by cAMP. They have also determined that cAMP induces several genes that are involved in the phosphoinositide signal transduction pathway (including PLD1) during decidualization, and they suggest that PLD1 plays a role in decidualization. In the present study, treatment of ES cells with 8-Br-cAMP upregulated PLD1 expression and PLD activity during decidualization, and treatment with PA for 3 days induced PRL expression and morphological change without 8-Br-cAMP treatment. Moreover, PLA2G1B blockage by pretreatment with mepacrine resulted in decreased PRL and IGFBP1 expression and morphological change. These data suggest that PA produced by PLD1 activation induces decidualization through PLA2G1B. PLA2G1B hydrolyzes the ester bonds of fatty acids at the sn-2 position of phospholipids, producing free fatty acids and lysophospholipids [28]. When the fatty acid is arachidonic acid, prostaglandin is generated, which acts as a multiple intracellular effector during decidualization. Kurusu et al. [29] have reported that arachidonyl trifluoromethyl ketone (ATK), which is a PLA2G1B inhibitor, causes dose-dependent inhibition of decidualization, and that PGE2 and prostacyclin have a weak effect in restoring ATK-inhibited decidualization. Therefore, they suggest that PLA2G1B induces decidualization through the generation of bioactive lipid mediators, such as prostaglandin (PG.). The importance of PGs in reproduction is evidenced by the multiple reproductive failures in mice when PG synthase 2 (PTGS2; COX-2) genes are disrupted [30]. Therefore, we suggest that PLD1 plays a role in the supply of bioactive lipid mediators, such as PG through PLA2G1B.
Rearrangement of the cytoskeleton is also important for morphological change during decidualization. Reorganization of stress fibers near the cell periphery has been reported in human ES cell that express the temperature-sensitive simian virus 40 large T antigen [31] and in a mouse decidualization model in vitro [32]. Furthermore, changes in the actin-based cytoskeleton and loss of focal adhesions within the decidualization-associated multicellular nodules have been reported [5]. Therefore, it is of a great interest to study whether the process of decidualization involves a protein that potentially regulates the focal adhesion assembly and organization of the actin cytoskeleton. Based on reports that PLD-dependent cytoskeletal rearrangements may play a pivotal role, it is tempting to suggest that PLD affects the actin cytoskeleton during decidualization. Iyer and Kusner [33] have reported that PLD activity is associated with the detergent-insoluble cytoskeleton. In support of this finding, Cross et al. have shown that actin stress fiber formation and PA accumulation in aortic endothelial cells can be inhibited by 1-butanol but not by 2-butanol [34]. In addition, Kam and Exton [35] have implicated PLD in the cross-linking of actin filaments mediated by -actinin. In agreement with this hypothesis, we observed that PLD upregulation induced morphological change in a PLD1-dependent manner, as shown by the selective inhibition conferred by DN-PLD1 transfection. Given the above considerations, it is highly likely that PLD1 is an essential factor for decidualization through its involvement in the reorganization of the cytoskeleton.
In conclusion, we have shown that PLD1 activity is increased during decidualization, and that PLD1 activation regulates the decidualization-specific phenotype through PLA2G1B. These characteristics suggest that PLD1 is one of the important regulators during decidualization, and that its activity affects successful implantation. Further studies to elucidate the signaling events that lead to PLD expression and eventual decidualization signaling events will broaden our understanding of the molecular mechanisms underlying decidualization and implantation.
FOOTNOTES
1Supported by the Korea Research Foundation (015-E00070, 2004) and partially supported by KOSEF and MOST through the Research Center for Biomedicinal Resources (Bio-Med RRC), Paichai University. 
Correspondence: 2Joong-Soo Han, Department of Biochemistry & Molecular Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791, Korea. FAX: 822 2294 6270; e-mail: jshan{at}hanyang.ac.kr
Received: 7 August 2006.
First decision: 28 August 2006.
Accepted: 23 October 2006.
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