Phosphatidic acid induces decidualization by stimulating Akt‐PP2A binding in human endometrial stromal cells

Decidualization of human endometrial stromal cells (hESCs) is crucial for successful uterine implantation and maintaining pregnancy. We previously reported that phospholipase D1 (PLD1) is required for cAMP‐induced decidualization of hESCs. However, the mechanism by which phosphatidic acid (PA), the product of PLD1 action, might regulate decidualization is not known. We confirmed that PA induced decidualization of hESCs by observing morphological changes and measuring increased levels of decidualization markers such as IGFBP1 and prolactin transcripts (P < 0.05). Treatment with PA reduced phosphorylation of Akt and consequently that of FoxO1, which led to the increased IGFBP1 and prolactin mRNA levels (P < 0.05). Conversely, PLD1 knockdown rescued Akt phosphorylation. Binding of PP2A and Akt increased in response to cAMP or PA, suggesting that their binding is directly responsible for the inactivation of Akt during decidualization. Consistent with this observation, treatment with okadaic acid, a PP2A inhibitor, also inhibited cAMP‐induced decidualization by blocking Akt dephosphorylation.


Introduction
For a successful pregnancy, the endometrium must grow continuously and mature throughout the menstrual cycle. Decidualization of human endometrial stromal cells (hESCs) is a crucial differentiation event characterized by biochemical and morphological alterations occurring from mid to late secretory phase of the menstrual cycle. During this process, decidual cells become rounded and enlarged, and this morphological change is accompanied by biochemical changes resulting in increased expression of several marker genes, including insulin-like growth factor-binding protein-1 (IGFBP1) and prolactin [1], and the progesterone level rises and intracellular cAMP increase dramatically. Based on these observations, the cAMP/PKA pathway has been extensively studied as the most likely cause of decidualization [2]. However, the molecular mechanisms underlying the decidualization process are not completely understood.
Phospholipase D (PLD) catalyzes the hydrolysis of phospholipids at the terminal phosphodiester bond. The most abundant phospholipid, phosphatidylcholine (PC), is hydrolyzed to phosphatidic acid (PA) and choline by activated PLD. PA regulates diverse intracellular signaling pathways as a lipid second messenger [3]. Moreover, it is metabolically converted to diacylglycerol (DAG) by lipin, which has phosphatidate phosphatase activity and to lysoPA or arachidonic acid by phospholipase A2 (PLA2). These metabolites also modulate various intracellular signaling processes [1,4]. In mammalian cells, two PLD isoforms (PLD1 and PLD2) have been cloned and characterized. Both have significant roles in processes such as proliferation, differentiation, vesicular trafficking, inflammation, and apoptosis and have been studied in the context of cancer, and neurodegenerative, cardiovascular, and infectious diseases [1,[5][6][7][8]. Arachidonic acid is involved in the cPLA2a/COX-2 pathway in hESCs [9] and the decidualization of uterine stromal cells [10] in vitro. Additionally, lysoPA regulates male and female reproduction [11], vascularization, and decidualization, which is related to embryo invasion during implantation [12]. However, the role of PLD1 or PA in the differentiation of hESCs is not fully understood.
Akt (protein kinase B, PKB) is a serine/threonine protein kinase, and is activated in a variety of cellular processes including apoptosis, proliferation, transcription, and metabolism [8,13,14]. The activity of the PI3K/Akt pathway is correlated with the decidualization progress [14,15] but its role is unknown. The transcription factor Forkhead box, class O1 (FoxO1) is directly regulated by Akt [16]. FoxO1 is a transcription factor, a member of the Forkhead box family [17]. During decidualization, the dephosphorylated form of FoxO1 is responsible for transcribing the IGFBP1 and prolactin genes [17,18]. But, how it is directly regulated in this context is not fully understood.
Protein phosphatase 2A (PP2A) is a conserved serine/threonine phosphatase with broad substrate specificity and diverse cellular functions, comprised of three subunits: a scaffold subunit (A), regulatory subunit (B), and catalytic subunit (C) [19,20]. PP2A in involved many cellular processes but was not known until now to play any role in the decidualization of hESCs.
The purpose of the present study was to see if PA, one of the enzymatic products of PLD1, regulates cAMP-induced decidualization of hESCs, and if so how acts. We found that phosphorylation of Akt and FoxO1 dramatically decreased in cells treated with cAMP or PA. Binding of PP2A to Akt also increased, suggesting that this was responsible for the dephosphorylation of Akt during decidualization. We therefore propose that PA promotes cAMP-induced decidualization in hESCs by stimulating interaction between PP2A and Akt, leading to inactivation of Akt and activation of FoxO1, which in turn is responsible for expressions of the decidualization-related genes.

PLD1 and PA are key regulators of the decidualization of hESCs
Previous studies have shown that PLD1 expression is up-regulated and PLD activity increases during the cAMP-induced decidualization of hESCs [1]. Therefore, we investigated whether loss-of-function of PLD1 affected the decidualization process. We induced decidualization by incubating cells in culture medium containing 0.5 mM 8-Br-cAMP (membrane-permeable cAMP analog) for 2 days; the hESCs developed a decidual-like morphology, becoming rounded with increased amounts of cytoplasm and enlarged nuclei (Fig. 1A). Moreover, the IGFBP1 and prolactin marker genes of decidualization were up-regulated (Fig. 1B), and these changes were abolished by treatment with the PLD inhibitor, FIPI (Fig. 1A,B). Furthermore, depletion of PLD1 transcripts with PLD1 siRNA gave similar results (Fig. 1C,D). To see whether phosphatidic acid (PA) induced decidualization, we treated hESCs with PA for 2 days in the absence of cAMP. PA treatment resulted in a decidual-like morphological change (Fig. 1E) as well as increased gene expression of IGFBP1 and prolactin (Fig. 1F). Collectively, these results suggest that PLD1 activation is an important process in decidualization of hESCs, and PA, the product of PLD1, acts as an essential signaling molecule.
such as FoxO1, ETS1, p53, STAT5, and C/EBPß are activated during decidualization [18,21,22]. FoxO1 is required for the expression of IGFPB-1 and prolactin [17,[23][24][25][26]. Under normal conditions, FoxO1 exists in a phosphorylated form and is present in the cytoplasm. When it is dephosphorylated by various factors, FoxO1 is translocated to the nucleus where it acts as a transcription factor [27,28]. As shown in Fig. 2A, a large percentage of FoxO1 protein existed in the phosphorylated form in control cells; the amount of the phosphorylated form was reduced by cAMP treatment and this reduction was antagonized by treatment with the PLD inhibitor FIPI ( Fig. 2A), or transfection of PLD1 siRNA (Fig. 2B). Moreover PA had the same effect as cAMP on FoxO1 dephosphorylation; when we added 50 lM or 100 lM PA for 40 min in the absence of cAMP, much of the FoxO1 became dephosphorylated, representing PA has same effect on the dephosphorylation of FoxO1 with cAMP (Fig. 2C). We also investigated whether the FoxO1 depletion affected decidualization ( Fig. 3A-C). As shown in Fig. 3B, the cAMP-induced decidual-like morphological change was completely disappeared by FoxO1 depletion using siRNA transfection. Decidualization marker genes (IGFBP1 and prolactin) were also drastically reduced by FoxO1 siRNA transfection ( Fig. 3C), which were consistent with previous studies [23][24][25][26]. Hence, these results indicate that PA, the product of PLD1, promotes FoxO1 dephosphorylation during cAMP-induced decidualization of hESCs.

PA induces Akt dephosphorylation during decidualization of hESCs
Many studies suggest that Akt is an upstream kinase in the phosphorylation of FoxO1 [29,30]. Moreover, Akt is dephosphorylated during decidualization [8]. We therefore tested the idea that PLD1 activation induces Akt dephosphorylation (and so renders it inactive) during decidualization thus protecting FoxO1 from being inactivated by phosphorylation by Akt. As expected, we found that phosphorylation of Akt on Thr308 during decidualization was decreased by cAMP, and PLD inhibition completely prevented the dephosphorylation of Akt in the presence of cAMP ( Fig. 4A), as did down-regulation of PLD1 with PLD1 siRNA (Fig. 4B). Next, to address the impact of PLD1 activation on Akt dephosphorylation at Thr308 during decidualization, we assessed whether PA mimics the cAMP-induced Akt dephoshorylation. Treatment with 50 lM or 100 lM PA for 40 min in the absence of cAMP mimicked cAMP in completely repressing Akt phosphorylation (Fig. 4C). Because Akt protein is activated by dual phosphorylation of Thr308 and Ser473, we also investigated the phosphorylation level of Akt on Ser473 during the decidualization. The pattern of phosphorylation on Ser473 was the same as that on Thr308 (data not shown). Taken together, these results demonstrate that PLD1 and its enzymatic product, PA, induce Akt dephosphorylation leading to inactivation of Akt during decidualization of hESCs.

PA induces FoxO1 dephosphorylation by inactivating Akt during decidualization
To see whether Akt inactivation is responsible for the dephosphorylation of FoxO1, we inhibited the Akt activity with the specific inhibitor, AKTi (Akt inhibitor) [31]. hESCs were incubated in culture medium containing 8-Br-cAMP or AKTi for 40 min. AKTi completely blocked the phosphorylation of FoxO1 showing the same effect on FoxO1 as cAMP alone (Fig. 5A). These results indicate that Akt is the kinase responsible for FoxO1 phosphorylation in hESCs.
Next, we examined whether Akt inhibition promoted decidualization in the absence of cAMP. As expected, hESCs treated with AKTi developed a decidua-like morphology reminiscent of the phenotypic response to cAMP (Fig. 5B). They also produced higher levels of IGFBP1 and prolactin transcripts (Fig. 5C). Taken together, these findings indicate that PLD1 activation leads to dephosphorylation of Akt, which in turn inhibits phosphorylation of FoxO1 and so activates it.

PP2A binding to Akt is responsible for the dephosphorylation of Akt/FoxO1 during decidualization
Akt inactivation is regulated by several phosphatases, such as PP2A and PHLPP. The former is a major serine/threonine phosphatase; it is regulated by interaction between its catalytic and regulatory subunits, and modulates the activity of various protein kinases such as Akt [19,32,33]. Moreover, PP2A is known to bind to PA in plant models [34,35]. Accordingly, we used the PP2A inhibitor, okadaic acid [19,36], to identify any effects of PP2A on PA-induced decidualization. Cells were pretreated with okadaic acid for 1 h and then differentiated with PA for 40 min. As shown in Fig. 6A, PA-induced Akt dephosphorylation at Thr308 was reversed by okadaic acid and phosphorylation of FoxO1 was increased (Fig. 6A). Furthermore, the development of decidua-like morphology was abolished (Fig. 6B), and expression of IGFBP1 and prolactin was also substantially decreased (Fig. 6C). This shows that inhibition of PP2A increases accumulation of phosphorylated Akt and the active Akt phosphorylates FoxO1, so inactivating it. Thus, PP2A seems to have an important role in decidualization by controlling Akt inactivation.
There is evidence that PP2A dephosphorylates Akt by binding to it [36]. To determine whether PP2A binds to Akt during decidualization, we performed pull-down assays after inducing decidualization by 8-Br-cAMP or PA. When cells were stimulated with 8-Br-cAMP or PA, PP2A was coimmunoprecipitated with Akt, indicating that the affinity of PP2A to Akt was increased by treatment with 8-Br-cAMP or PA (Fig. 6D). Taken together, these results suggest that during decidualization, PA promotes the formation of an Akt-PP2A binding complex, and Akt dephosphorylation then leads to increased dephosphorylation (and activation) of FoxO1 (Fig. 6E).

Discussion
Decidualization is a process characterized by dramatic changes in the biochemical properties as well as morphological appearance of hESCs required to establish implantation and to maintain pregnancy [37,38]. Progesterone and cAMP are decidualization inducers [39,40]. Previously, we showed that PLD1 activity and expression increase during cAMP-induced decidualization [1,41]. Additionally, we found that PLD1 expression is up-regulated by the progesterone receptor, which acts as a transcription factor for PLD1 during decidualization [42]. However, the mechanisms by which PLD1 regulates decidualization remained unknown. In the present work, we confirmed the role of PLD1 in decidualization; cellular transformation into a decidual-like morphology by cAMP was completely inhibited by PLD1 inhibition or mRNA depletion (Fig. 1A,C). Similarly, increased expressions of IGFBP1 and prolactin during decidualization were significantly repressed by loss-of-function of PLD1 (Fig. 1B,D). Furthermore, we showed that PA, an enzymatic product of PLD1, mimics the effects of PLD1 on decidualization. FoxO1 plays a major role as a transcription factor in IGFPB-1 and prolactin gene expression during decidualization of hESCs [17]. In mammalian cells, FoxO1 activity is inhibited by phosphorylation of Ser256 by Akt, and phosphorylated FoxO1 translocates from the nucleus to the cytosol [16]. The F-box protein, SKP2, and E3 ubiquitin ligases recognize phosphorylated FoxO1 and FoxO1 is slowly degraded by the ubiquitin-proteasomal system [43]. We found that FoxO1 activation (dephosphorylation) was elevated when we treated hESCs with cAMP or PA (Fig. 2), implicating that it acts as a transcription factor in cAMP-or PA-induced decidualization. Furthermore, several reports have suggested that Akt activity is regulated by progesterone and/or cAMP treatment [8,14]. Accordingly, we found that Akt activity was regulated by cAMP or PA (Fig. 4), and was responsible for FoxO1 phosphorylation in hESCs (Fig. 5).
PP2A is a representative serine/threonine phosphatase, and targets various kinases, like Akt [44]. We demonstrated that PA promotes cAMP-induced decidualization in hESCs by inactivating Akt via an Akt-PP2A complex (Fig. 6). However, as shown in Fig. 6, PA-induced decidualization was not completely inhibited by the PP2A inhibitor, okadaic acid. This indicates that not only PP2A but also some other phosphatase(s) is involved in the PA-induced Akt inactivation. Indeed, PHLPP, another major serine/threonine phosphatase, selectively dephosphorylates Ser473 in the hydrophobic motif at the C terminus of Akt [33,45]. Therefore, we suspect that PHLPP also induces Akt dephosphorylation on Ser473 in differentiating hESCs.
In addition to these phosphatases, ribosomal protein S6 kinase 1 (S6K1) and insulin receptor substrate 1 (IRS1) also regulate Akt phosphorylation. Insulin binds to the insulin receptor on the plasma membrane and triggers an increase in tyrosine phosphorylation of IRS1 [46]. Moreover, the IRS1-associated class 1 PI3K recruits Akt to the plasma membrane by increasing PIP 3 production [47]. In this case, Akt is phosphorylated at Thr308 by PDK1. The mammalian target of rapamycin (mTOR)/S6K1 pathway mediates several biological effects of nutrients, insulin, and energy, and is activated by insulin. The rapamycin-insensitive companion of mTOR (rictor) complex 2 (mTORC2) mediates Akt phosphorylation at Ser473. Akt is simultaneously phosphorylated by mTORC2 and PDK1 at Ser473 and Thr308 [48]. On the other hand, the rapamycin-sensitive companion of mTOR (raptor) complex 1 (mTOR1) regulates S6K1 phosphorylation at a number of residues in an Akt-independent pathway [49]. Interestingly, under nutrient overload conditions, mTORC1/S6K signaling is negatively regulated in insulin signaling through inhibition of IRS1 transcription and IRS1 phosphorylation at a serine residue [46]. Moreover, the mTORC2/S6K1 pathway represses IRS1, and Akt is subsequently inactivated by this negative pathway [46]. These reports indicate that PA may also control mTOR/S6K1 signaling by inactivating Akt. Accordingly, we suggest that the PP2A and the IRS1 pathway through S6K1 may jointly mediate Akt dephosphorylation in decidualization models. We also considered the possibility that cAMPregulated guanine nucleotide exchange factors (Epac) might be involved in FoxO1 dephosphorylation in our system. cAMP stimulates EPAC by directly binding to PA in the plasma membrane [50]. Moreover, the Epac/ Rap1 signaling pathway is activated during decidualization of hESCs, and FoxO1 phosphorylation is regulated through the Epac/ERK pathway in the same models [51]. Thus, these reports suggest that the association between PA and EPAC can increase FoxO1 activity during decidualization in hESCs.
Aberrant decidualization elicits physiological dysfunction of the endometrium and is associated with pathologies such as endometriosis, deciduosis, and endometrial cancer [52][53][54]. Endometrial decidualization converts the normal endometrium into a specialized uterine lining adequate for optimal accommodation of the gestation [55]. The presence of endometrial gland and stroma outside of endometrial cavity induces endometriosis [56], which affects 10-15% of women at reproductive age and is associated with plevic pain and infertility [57]. Moreover, endometriotic stromal cells lose their capacity for cellular differentiation in the ectopic environment [58]. Furthermore, decidualized tissue can grow in ectopic sites during pregnancy, resulting in deciduosis with a gross appearance that macroscopically mimics a malignant tumor [53]. In present study, we found that PA controls decidualization by dampening Akt-FoxO1 signaling pathway, which was observed in endometriosis and endometrial cancer patients group [59][60][61][62]. In this context, we are tempting to conclude that PA might be implicated in various endometrial disorders, such as deciduosis and endometriosis and subsequently fertility.
In spite of the clinical importance of decidualization, the mechanisms of decidualization are not clearly defined in vivo due to its complexity. Hence, in vitro systems of decidualization have been well established to overcome some of the inherent limitations of studying [63]. Nevertheless, several factors of in vitro system restricts the importance of studies, such as concern of artificial system, cellular variation of isolated stromal cells, and effect of cultural environment on in vitro decidualization [64,65]. Future study will need to examine the role of PA in in vivo decidualization systems using either pregnant or pseudopregnant mice through uterine injection of oil or sepharose beads or mechanical stimulation [66,67].
In conclusion, the present study demonstrated that PA is a critical mediator in cAMP-induced decidualization of hESCs through regulating direct binding between PP2A and Akt.

Ethics statement
Samples were collected under protocols approved by the Institutional Review Board of Hanyang University Hospital, and written informed consent was obtained from all participants.    surgery for nonendometrial abnormalities in Hanyang University Hospital between September 2008 and September 2014. Each endometrial specimen obtained was examined histologically. hESCs were isolated as described previously. Briefly, tissue samples were collected in DMEM containing 100 IUÁmL À1 penicillin, 100 lgÁmL À1 streptomycin, 2 mM L-glutamine, and 10% (v/v) FBS. After cleaning and trimming to remove blood clots and mucus, the specimens were minced to fragments of less than 1 mm in size under a laminar flow hood and digested at 37°C for 60 min with 0.25% collagenase I. The cell suspension was filtered twice through a 40 lm pore-size sieve (BD Falcon, Bedford, MA, USA). After enzymatic digestion, most of the stromal cells were present as single cells or small aggregates. The purity of the stromal cells obtained by this method was typically > 90%, as determined by immunocytochemical staining for vimentin, a stromal cell marker. The purified stromal cells were washed, and viable cells were counted by dye exclusion using trypan blue. The viability of the isolated cells was at least 90% in each experiment. hESCs were cultured to confluence in 100-mmdiameter culture dishes at 37°C in DMEM supplemented with 10% (v/v) FBS, 100 IUÁmL À1 penicillin, and 100 lgÁmL À1 streptomycin in a humidified atmosphere of 5% CO 2 in air. To induce in vitro decidualization, cells were exposed to 0.5 mM 8-Br-cAMP for 3 days. Phasecontrast microscopy was used to verify the morphological changes associated with differentiation.

Statistical analysis
Data are expressed as means AE SEM of at least five independent experiments; P < 0.05 was considered statistically significant. Comparisons between groups were made using unpaired Student's t-tests using the statistical package GRAPHPAD PRISM 6 (GraphPad Software Incorporated, La Jolla, CA, USA).