Functional Analysis of p21Cip1/CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

Preeclampsia (PE), a gestational hypertensive disease originating from the placenta, is characterized by an imbalance of various cellular processes. The cell cycle regulator p21Cip1/CDKN1A (p21) and its family members p27 and p57 regulate signaling pathways fundamental to placental development. The aim of the present study was to enlighten the individual roles of these cell cycle regulators in placental development and their molecular involvement in the pathogenesis of PE. The expression and localization of p21, phospho-p21 (Thr-145), p27, and p57 was immunohistochemically analyzed in placental tissues from patients with early-onset PE, early-onset PE complicated by the HELLP (hemolysis, elevated liver enzymes and low platelet count) syndrome as well as late-onset PE compared to their corresponding control tissues from well-matched women undergoing caesarean sections. The gene level was evaluated using real-time quantitative PCR. We demonstrate that the delivery mode strongly influenced placental gene expression, especially for CDKN1A (p21) and CDKN1B (p27), which were significantly upregulated in response to labor. Cell cycle regulators were highly expressed in first trimester placentas and impacted by hypoxic conditions. In support of these observations, p21 protein was abundant in trophoblast organoids and hypoxia reduced its gene expression. Microarray analysis of the trophoblastic BeWo cell line depleted of p21 revealed various interesting candidate genes and signaling pathways for the fusion process. The level of p21 was reduced in fusing cytotrophoblasts in early-onset PE placentas and depletion of p21 led to reduced expression of fusion-related genes such as syncytin-2 and human chorionic gonadotropin (β-hCG), which adversely affected the fusion capability of trophoblastic cells. These data highlight that cell cycle regulators are important for the development of the placenta. Interfering with p21 influences multiple pathways related to the pathogenesis of PE.


Introduction
Pregnancy can be affected by various health problems, of which preeclampsia (PE) is the most common. PE is a multisystemic gestational disease with a global prevalence of up to 8% [1]. It is characterized by concurrent hypertension and proteinuria or any other sign of end organ damage including liver or brain, occurring after 20 weeks of gestation [2]. PE is a consequence of diverse pathophysiological processes linked to maternal endothelial dysfunction and systemic inflammation, which can result in multiorgan failure, if the fetus and placenta are not delivered [1][2][3]. It can be subdivided into an early-onset (<34th week This study was approved by the Ethics Committee at the University Hospital (reference number: 375/11), Goethe-University Frankfurt. Written informed approval was obtained from all donors. PE was diagnosed as an occurrence of hypertension after 20 weeks of gestation with a blood pressure ≥140/90 mmHg and proteinuria with ≥300 mg in 24 h. The HELLP syndrome was defined as the presence of hemolysis, elevated liver enzymes, and thrombocytopenia (low platelet count <100,000/µL). The reasons for delivery of earlyonset control groups were PE-irrelevant like breech presentation, premature rupture of membranes, premature placental abruption, non-reassuring fetal heart rate, or umbilical cord prolapse. Tissue samples were taken from placentas within 30 min post-delivery, formalin-fixed and paraffin-embedded (FFPE) for immunohistochemistry staining (IHC), or frozen immediately in liquid nitrogen for mRNA and protein extraction, which were stored at −80 • C until usage. Clinical information of all participants is shown in (Tables 1-4). For IHC staining, Prof. Dr. Qi Chen, Department of Obstetrics and Gynecology, University of Auckland, and Fudan University, Shanghai, China, kindly provided us with six first trimester placental FFPE samples (six to nine weeks of gestation, age 20 to 33 years). The sample collection was approved by the Ethics Committee of the Hospital of Obstetrics and Gynecology of Fudan University (reference number 2018-62), China. Written consent was obtained from healthy donors undergoing elective surgical terminations of pregnancy. For trophoblast organoid formation, primary villous cytotrophoblasts or placental mesenchymal stem/stromal cells were isolated from first trimester placentas. Written informed consent was obtained from patients undergoing elective terminations of normal pregnancies (seven to 12 weeks of gestation, age 25 to 41 years) at the Medical Practice for gynecology and obstetrics led by Dr. Thorsten Nowak with ethical approval from the Ethics Committee at the University Hospital, Goethe-University Frankfurt (reference number .
Trophoblast fusion was induced by treating cells with 25 µM forskolin (Sigma-Aldrich, Taufkirchen, Germany) for indicated time points. An equal amount of dimethyl sulfoxide (DMSO, Sigma-Aldrich, Taufkirchen, Germany) was used as the vehicle control.

Microarray Analysis
BeWo cells were treated with control siRNA or siRNA targeting the UTR of p21 (sip21) for 48 h. Cells from three independent experiments were harvested and the total RNA was isolated using RNeasy Kits (Qiagen, Hilden, Germany). The expression was assessed using Human HT-12 v4 Beadchip (Illumina, San Diego, CA, USA), a direct hybridization whole-gene expression array. The expression profiling service from the German Cancer Research Center (DKFZ Microarray Core Facility, Heidelberg, Germany) was used. The most significant genes with a p-value (Student's t-test) less than 0.05 were selected. A full gene list is available as Supplementary Table S1, which shows data from the whole gene-expression array with a p-value smaller than 0.05.

Statistical Analysis
Outliers were detected with Grubbs' test (GraphPath QuickCalcs, San Diego, CA, USA). Data distribution normality was analyzed with the Shapiro-Wilk test and statistical significance was analyzed with the Student's t test, or, if not Gaussian distributed, with the non-parametric Wilcoxon-test (paired) or the Mann-Whitney U test (unpaired samples). Difference was defined as statistically significant when p < 0.05.

Cell Cycle Regulators Are Affected by the Delivery Mode, and Specifically Expressed in Trophoblast Organoids and Placental Tissues
The rapid expansion of the human placenta is attributed to spatiotemporally regulated cell proliferation, for which the cell cycle regulators are indispensable. A previous study showed that the mode of delivery affected the expression of certain genes including CDKN1C (p57), which was elevated over 2-fold in placental tissues from labor deliveries compared to elective caesarean sections [44]. We started to verify and extend this very important issue in terms of the impact of the delivery mode on gene expression of CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57). Placental tissues were collected from healthy women with different delivery modes without significant differences in gestational age, maternal age, and maternal body mass index (BMI) ( Table 1). Total RNAs were extracted for gene analysis. We compared the gene expression levels of placental tissues from elective caesarean section (CS), emergency caesarean section after the onset of labor (eCS), vaginal delivery (VD), and operative vaginal delivery (opVD, mainly forceps delivery). Indeed, as shown in Figure 1A, the delivery mode strongly affected the gene expression of cell cycle regulators in the human placenta, especially for CDKN1A and CDKN1B, where a significant increase of about 2-fold was obtained after VD and opVD delivery. For CDKN1C, a significant 2-fold elevation was observed by opVD. Based on these data, we decided to collect placental tissues from caesarean sections without mechanical compression caused by uterine contractions or additional stress factors. Cells 2021, 10, x FOR PEER REVIEW 9 of 25  To gain insight into the possible roles of cell cycle regulators in placental development, we generated human trophoblast 3D organoids (ORGs) according to well-established protocols [26,28,29]. Villous cytotrophoblasts (CTBs) were purified from first trimester placental tissues and embedded in Matrigel or plated on ultra-low attachment plates ( Figure S1A). The trophoblast origin was corroborated by immunohistochemistry-immunofluorescence (IHC-IF) staining of the presence of the trophoblast marker cytokeratin 7 and epidermal growth factor receptor (EGFR), the proliferation marker Ki67, and the absence of human leukocyte antigen G (HLA-G) (Figure S1B), and by analyzing the gene expression of GATA3 (GATA binding protein 3), TFAP2A (transcription factor AP-2 alpha), TFAP2C (transcription factor AP-2 gamma), and ELF5 (E74 like ETS transcription factor 5) (Figure S1C), previously described trophoblast identity criteria [28]. The data revealed similar gene expression profiles between first trimester organoids (ORGs) and isolated primary cytotrophoblasts (pCTBs); both displayed higher expression levels of GATA3, TFAP2A, TFAP2C, and ELF5 compared to placental mesenchymal stem/stromal cells (pMSCs) isolated from first trimester placental tissues. Additionally, relative to first trimester pCTBs, trophoblast organoids expressed very high levels of CGbeta5 (β-hCG) ( Figure S1D). IHC-IF staining for cytokeratin 7 revealed that organoids had the previously described inside-out structure [28,29], where the CTBs formed the outer layer and fused toward the center to generate the STB ( Figure 1B and Figure S1B). In comparison, the IHC-IF of first trimester placenta showed the familiar structure ( Figure 1C). Interestingly, in both trophoblast organoids and placental tissue sections, p21 was expressed in CTBs and the STB ( Figure 1B,C), in the nucleus and the cytoplasm.
For further analyses, we used sections of placental tissues with gestational ages between six and nine weeks, between 25 and 33 weeks as well as between 34 and 40 weeks from normal donors. Using IHC, placental sections were stained for p21, p-p21 (Thr-145), p27 or p57, and counterstained with hematoxylin. Whereas p21's role in cell cycle arrest is attributed to its nuclear localization, its phosphorylation at Thr-145 by distinct kinases is described as a marker for cytoplasmic translocation or enhanced protein stability contributing to cell cycle progression [23]. Placental tissue was highly positive for p21, p-p21, and p27 ( Figure 1E). The positive staining was found in the cytoplasm as well as the nucleus of trophoblastic cells of the placenta, especially in the proliferative villous CTBs, in particular, CTBs ongoing to fuse to the STB, so called fCTBs marked by partial loss of E-cadherin staining, suggesting the breakdown of apical and lateral plasma membranes (fCTBs, Figure 1D), the terminally differentiated, non-proliferative, and multinucleated STB, the migrating EVTs in proliferative cell columns, and villous stromal cells throughout gestation. The positive staining of p57 was predominantly present in the nucleus of fCTBs, stromal cells, and EVTs ( Figure 1E, last panel).

Cell Cycle Regulators Are Highly Expressed during the First Trimester of Gestation
We analyzed the positively stained CTBs, fCTBs, and the STB area for p21, p-p21, p27, and p57 in tissue sections from healthy donors with gestational ages between six and nine weeks (n = 6), between 25 and 33 weeks (n = 20; Table 2, also served as early-onset control group), and between 34 and 40 weeks (n = 10; Table 3, also served as late-onset control group) (representatives are shown in Figure 1E). In the first trimester sections, there were not enough EVTs for a reliable quantification. For all staining, first trimester sections showed the highest percentage of positive CTBs and positive STB area with a significant decline in the early-and late-onset control group (Figure 2A,B; named 25-33 or 34-40 week). There was a significant difference in p-p21 positive staining of CTBs and the STB area between early-and late-onset control samples (Figure 2A,B, middle panel). Interestingly, fCTBs displayed high percentages of positive staining of p21, p-p21, p27, and p57 throughout gestation ( Figure 2C), suggesting that this cell population is highly active in proliferation and differentiation. Next, the gene expression of CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57) was evaluated in placental tissues from the first trimester samples (named 6-9 week) compared to early-and late-onset controls ( Figure 2D). The relative amounts of CDKN1A, CDKN1B, and CDKN1C in early-onset controls were significantly reduced by 92%, 79%, and 90%, respectively, compared to the first trimester group. Particularly, CDKN1A was significantly reduced in early-as well as late-onset controls compared to the first trimester samples ( Figure 2D, left). In addition, a moderate increase was observed in the gene expression of all cell cycle regulators in late-onset controls compared to early-onset controls ( Figure 2D).

p21 Expression Is Reduced in fCTBs of Early-Onset PE Placental Samples
To address if cell cycle regulators were altered in preeclamptic placentas, the expression levels of p21, p-p21, p27, or p57 were compared between twenty early-onset PE placental tissues and twenty samples from well-matched control donors (Table 2) by the semi-quantitative H-score method, which combines the percentage of stained cells/area and their staining intensity. There was no apparent difference in cell cycle regulators in the H-score of CTBs or the STB, and the percentage of positive CTBs or in the positive stained area per visual field in the STB in early-onset PE samples compared to their respective control counterparts ( Figure 3A,B). Interestingly, a significant reduction of p21 in the Hscore of fCTBs as well as in the percentage of positive fCTBs were observed in early-onset preeclamptic placental samples, in comparison to the matched control tissues ( Figure 3C, left graph). While p-p21 and p27 were almost comparable ( Figure 3C, 2nd and 3rd graph), the percentage of positive p57 fCTBs declined in early-onset PE samples compared to the control tissues ( Figure 3C, right graph, bottom). Moreover, the percentage of p57 positive stained EVTs was significantly reduced, whereas p21, p-p21, and p27 were hardly altered ( Figure 3D). To underscore these results, we next evaluated the expression with western blot analyses from whole tissue samples. While the expression of p21, p27, and p57 was decreased, only p57 showed a significant reduction ( Figure 3E). A limitation of our study is that the p-p21 antibody did not work for tissue western blot analysis. Further gene analysis showed reduced levels of CDKN1A (p21) and CDKN1B (p27) ( Figure 3F). onset controls ( Figure 2D). The relative amounts of CDKN1A, CDKN1B, and CDKN1C in early-onset controls were significantly reduced by 92%, 79%, and 90%, respectively, compared to the first trimester group. Particularly, CDKN1A was significantly reduced in early-as well as late-onset controls compared to the first trimester samples (Figure 2D  left). In addition, a moderate increase was observed in the gene expression of all cell cycle regulators in late-onset controls compared to early-onset controls ( Figure 2D). . The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Unpaired Student's t-test or Mann-Whitney U test referring to first trimester samples was used for statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast.  TBP was used as the endogenous control. Paired Student's t-test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Reduced p21 Protein Expression in Early-Onset PE with HELLP Syndrome
Ten late-onset PE samples and ten control tissues from well-matched donors (Table 3) were also systematically analyzed. Obvious change in p21, p-p21, p27, and p57 expression was not observed at protein as well as at gene level ( Figure S2). Interestingly, the H-score of p-p21 was significantly elevated in the STB ( Figure S2B, middle graph). In sum, these data support the notion that early-and late-onset PE derive from distinct pathogenesis and should be considered separately from each other.

Reduced p21 Protein Expression in Early-Onset PE with HELLP Syndrome
Since early-onset PE is often complicated by the HELLP syndrome (ePEH) [1], placental samples were also collected from patients with ePEH and their well-matched controls ( Table 4). The percentage of positive cells/area and the staining intensity of CTBs, the STB, and fCTBs were evaluated for p21, p-p21, p27, and p57 ( Figure 4A-C). There was no obvious difference of cell cycle regulators in the H-score of CTBs or the STB, and the percentage of positive CTBs or in the positive stained area of the STB in ePEH compared to the well-matched controls ( Figure 4A,B). Interestingly, there was a significant increase in the H-score of p-p21 in fCTBs ( Figure 4C). The percentage of positive stained EVTs was comparable ( Figure 4D). Western blot analyses with cellular lysates from whole tissue samples showed that the expression of p21, p27, and p57 was decreased, and only p21 showed a significant reduction ( Figure 4E). Further gene analysis showed reduced levels of CDKN1A (p21) and CDKN1B (p27) (Figure 4F), as observed in early-onset PE ( Figure 3F).

p21 Expression Is Decreased in Trophoblastic Cell Lines and in Isolated Primary Cytotrophoblasts under Hypoxic Conditions
PE is associated with chronic hypoxia of the placenta through defective trophoblast invasion and inadequate remodeling of the maternal spiral arteries [5,17,45,46]. To mimic the situation in PE, immortalized first trimester trophoblast cell lines SGHPL-4 [40] and HTR [39] were grown under normal (21.4% O 2 ) or hypoxic conditions (1% O 2 ) for 48 h. The gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53), the master regulator of p21, were measured. CDKN1A was significantly reduced under hypoxic conditions in SGHPL-4 cells, whereas CDKN1B, CDKN1C, and TP53 were decreased but not significantly ( Figure 5A). Moreover, the p21 protein was also reduced, while the p53 protein expression was not affected by hypoxia ( Figure 5B). Comparable results were also observed with HTR cells, which showed a significantly lowered gene expression of CDKN1A and CDKN1B ( Figure 5C) as well as decreased p21 protein expression under hypoxic conditions ( Figure 5D). To investigate cell cycle regulators in a more physiological setting, trophoblast organoids (ORGs) derived from first trimester placenta were generated and cultured under normal (21.4% O 2 ) or under hypoxic conditions (1% O 2 ) for 48 h. The expression of cell cycle regulator genes was also affected, showing a significant decline in CDKN1A, CDKN1B, CDKN1C, and TP53 under hypoxia ( Figure 5E). The choriocarcinoma cell line BeWo, a widely used cell culture model mimicking CTB fusion and differentiation [47], was also grown under normoxia and hypoxia for gene analysis. CDKN1A, CDKN1B, and TP53 were significantly reduced under low oxygen supply ( Figure 5F).
Since early-onset PE is often complicated by the HELLP syndrome (ePEH) [1], placental samples were also collected from patients with ePEH and their well-matched controls ( Table 4). The percentage of positive cells/area and the staining intensity of CTBs, the STB, and fCTBs were evaluated for p21, p-p21, p27, and p57 proteins ( Figure 4A-C). There was no obvious difference of cell cycle regulators in the H-score of CTBs or the STB, and the percentage of positive CTBs or in the positive stained area of the STB in ePEH compared to the well-matched controls ( Figure 4A,B).   To further underline the observations, human primary cytotrophoblasts (pCTBs) were isolated from five term placentas of healthy donors, which were grown under normoxia or hypoxia and compared to pCTBs isolated from late-onset PE patients grown under normoxic conditions (Table 5). The characterization of isolated trophoblastic cells was performed with positive staining of EGFR, E-cadherin, β-hCG, cytokeratin 7, cytokeratin 18, and negative markers HLA-G and vimentin ( Figure S3). Compared to normoxia, the gene levels of CDKN1A (p21), CDKN1B (p27), and TP53 (p53) were significantly reduced in primary cytotrophoblasts under hypoxic conditions ( Figure 5G). Interestingly, a decrease in CDKN1A and CDKN1B expression was also observed in the pCTBs from late-onset PE placentas, albeit not significant due to the small sample size ( Figure 5G). Table 5. Clinical information of preeclamptic patients and matched donors, whose placentas were used for primary trophoblast isolation (term). Mean value ± standard deviation is shown. BP, blood pressure; n.d., not determined.

Knockdown of p21 Impairs the Fusion Ability of Trophoblastic BeWo Cells
Since the expression of p21 was decreased in fCTBs, a cell population ready to fuse into the STB of placental tissues from early-onset PE patients ( Figure 3C, left graph), we focused on the role of p21 in cell differentiation and fusion. To look at possible mechanisms by which reduced p21 could cause defects in cell differentiation, total RNAs were extracted from BeWo cells depleted of p21 for RNA microarray analysis ( Figure 6A, Table S1). Differently expressed genes were compared between BeWo cells treated with the control siRNA (sicon) and siRNA specifically targeting p21. The heatmap depicts genes with a p-value smaller than 0.05, and a fold change greater than 1 (red color code) and below 1 (blue color code), respectively ( Figure 6A, left panel).
Interestingly, as reported for HTR cells depleted of p21 [25], the extracellular signalregulated kinase 3 (ERK3), encoded by the gene MAPK6, is among the top three of altered genes and strongly reduced upon p21-depletion (hit number 1, CDKN1A). ERK3 is a distantly related member of the mitogen-activated protein kinase (MAPK) superfamily [48], which is known to be involved in cell differentiation [49]. Further analysis revealed that the fusion-related genes HERV-FRD (syncytin-2), ERVWE1 (syncytin-1), CGbeta5 (β-hCG), KLF6 (Krüppel-like factor 6), and GCM1 (glial cells missing transcription factor 1) were reduced ( Figure 6A, right upper panel) upon p21 depletion ( Figure 6A, right lower panel). This was corroborated for HERV-FRD, CGbeta5, and GCM1 by quantitative PCR analysis with BeWo ( Figure 6B) and JEG-3 cells ( Figure 6C). To further address this issue, BeWo or JEG-3 cells depleted of p21 with two different siRNAs (sip21 and sip21 #2; Figures 6F and S3G) were stimulated to fuse with forskolin up to 48 h. The amount of β-hCG, which is upregulated upon cell fusion and induced by forskolin [47], was reduced upon p21 depletion, visualized by immunofluorescence staining compared to the control cells (Figures 6D and S3E). Moreover, the luciferase assay showed that reduced p21 significantly decreased the expression of syncytin-2 in a promoter-dependent manner in BeWo and JEG-3 cells, respectively ( Figures 6E and S3F).

Discussion
The cell cycle regulator p21 is a key player in various cellular events including cell differentiation, migration, stem cell maintenance, and gene transcription [24], processes important for placental development and altered in its pathogenesis. However, p21's expression and roles in the placenta and its diseases are still contradictory, despite numerous studies and intensive work. To clarify these issues, in the present work, we collected early-onset PE, early-onset PE complicated by the HELLP syndrome, and late-onset PE placental samples, and their well-matched control tissues, and systematically examined the expression of p21 (CDKN1A) and its family members p27 (CDKN1B) and p57 (CDKN1C) as well as their roles in normal pregnancy and hypertensive disorders. We show here that these cell cycle regulators are highly expressed in first trimester placentas, while their expression generally decreases in the second and third trimester placenta at the protein as well as gene level. Notably, fCTBs, a special portion of CTBs ongoing to fuse into the STB, express high levels of p21 and its family members, which remain high throughout gestation. Importantly, the level of p21 is only reduced in fCTBs of early-onset PE placental tissues and its overall protein expression is decreased in early-onset PE complicated by the HELLP syndrome. These data strongly support the notion that early-and late-onset PE derive from distinct pathogenesis. Moreover, cell cycle regulators are decreased upon hypoxic conditions and depletion of p21 leads to reduced expression of fusion-related genes and an impaired fusion capacity of trophoblastic cells. In addition, the expression of these cell cycle regulators is dependent on the delivery mode, underscoring the importance of optimization and standardization of patient selection and placental collection for proper interpretation of existing data on cell cycle regulators.
We show that overall p21 was reduced in early-onset PE and early-onset PE complicated by the HELLP syndrome, whereas it was unaltered in late-onset PE, suggesting its potential involvement in the development of early-onset PE. Several studies provide insight into p21 s roles in PE and are, however, accompanied by inconsistency and divergence: one study reported that the gene and protein expression of p21 was increased in preeclamptic placentas [50], whereby another study showed that p21 expression was decreased via IHC staining [51]. In other investigations, its protein or gene expression was unchanged [52,53]. The contradictory expression of p21 in PE might be explained by the highly variable study design concerning gestational age without the differentiation in early-and late-onset PE or concomitant diseases like the HELLP syndrome, sample number, and mode of delivery. In further support of our results, a recent study showed a decrease in p21 in early-onset PE, whereas it was unchanged in late-onset PE at the gene and protein level [54]. The reduced p21 is proposed to lead to elevated Cdk2/cyclin E levels and reduced phospho-retinoblastoma protein (RB) critical for cell cycle progression, which hampered the differentiation and fusion process of CTBs to the STB [54].
PE, especially early-onset PE, is associated with constant hypoxia and oxidative stress of the placenta [17]. Interestingly, we show that hypoxia significantly decreased mRNA levels of CDKN1A (p21) and CDKN1B (p27) in diverse trophoblastic cell lines, organoids derived from first trimester placenta, and isolated primary cytotrophoblasts. Others reported that the p21 protein was downregulated in CTBs upon 2% oxygen supply for 72 h, which was 3.8-fold higher in normoxia cell extracts isolated from explants of anchoring villi of first trimester placenta [55]. Moreover, we observed that hypoxia also decreased the mRNA level of TP53 (p53), the upstream regulator of p21. However, the p53 protein expression from whole cellular lysates was not affected. p53 downregulation was reported in the STB of primary isolated term trophoblasts upon 1% hypoxia for 24 h, associated with the induction of apoptosis [56]. Evaluation of nuclear p53 and its post-modifications is required to clarify whether p53 is responsible for reduced p21. Interestingly, transcriptional repressors of p21 like BCL6 (B cell lymphoma 6) [57] or TFAP2C [58] are known to be enhanced in PE [52,59]. In addition, the miRNA family miR-130 including miR-130a/b and miR-301a/b repressed CDKN1A in human pulmonary artery smooth muscle cells upon hypoxia [60]. Interestingly, miR-130a and miR-301a have been reported to be overexpressed in PE placenta [61]. These regulators may be responsible for the reduced expression of p21 under hypoxia, bridging its reduction to the pathogenesis of PE.
PE is associated with profound cellular dysfunctions including reduced differentiation and fusion ability [62], and p21 is of crucial importance for differentiation [24]. Indeed, p21 protein levels increased during spontaneous differentiation and fusion of term CTBs [63] as well as CDKN1A during differentiation of mouse trophoblast stem cells and fusion of BeWo cells upon forskolin treatment [64], whereas p53 levels were reduced during BeWo cell differentiation [65]. We report here that p21 levels were significantly decreased in fCTBs, a special portion of CTBs ongoing to fuse to the STB, in early-onset PE samples, suggestive of its involvement in CTB differentiation and fusion. Moreover, in early-onset PE complicated by the HELLP syndrome, the H-score of overall p21 was reduced, whereas phosphorylated-p21 was significantly elevated in fCTBs, possibly a compensatory mechanism in a crisis situation induced by the HELLP syndrome, by which the stress protein p21 is trying to be stabilized and possibly translocated to the cytoplasm, where it could exert its antiapoptotic function but hardly affect the transcription of fusion-related genes. In fact, our data revealed that the fusion-related genes HERV-FRD (syncytin-2), CGbeta5 (β-hCG), and GCM1 were reduced upon p21 depletion. Importantly, decreased p21 significantly reduced the expression of syncytin-2 in a promoter-dependent manner in BeWo and JEG-3 cells. Indeed, p21 has been reported to interact as a co-activator with GCM1 to bind to the promoter region of syncytin-2, regulating its transcription [66]. Our findings are further supported by previous studies. Reduced p21 was reported to be involved in impaired fusion mediated by KLF6 silencing in BeWo cells [63]. Recently, it has been revealed that p21, but not its family member p27, coordinates trophoblast fusion contributing to G0 arrest and terminal differentiation [66]. Of significance, the reduced expression of the fusion-related genes/proteins syncytin-1 and syncytin-2 correlates with the severity of PE [67]. In sum, our data clearly suggest that reduced p21 in fCTBs compromises the expression of fusion-related genes, contributing to impaired differentiation and fusion of trophoblasts, an important hallmark of early-onset PE.
The studies concerning p27 and p57 in PE are rather limited: in one study, p27 and p57 were significantly increased in preeclamptic placentas [68]. However, in that study, the averaged gestational age of PE patients was lower than in the control group [68]. p57 has been reported to be important in trophoblast fusion [69,70], and in migration and invasion [71]. In early-onset PE samples, we observed a significant reduction in the percentage of p57 positive fCTBs, suggestive of its potential contribution to compromised CTB differentiation and fusion, possibly in collaboration with reduced p21, in early-onset PE placentas. Additionally, we detected a reduced p57 level in EVTs, indicative of a possible involvement in impaired cell motility, a hallmark of PE. In fact, the amount of p57 was also decreased in western blot analyses of early-onset PE placental samples, whereas we did not observe significant differences in p27 expression. Interestingly, mutant mice, defective only for the maternal p57 allele, displayed clinical manifestations of PE including proteinuria and elevated blood pressure [72]. However, a further study failed to reproduce these PE manifestations using the same mouse model, although mice exhibited placental abnormalities [73]. p57-mutant murine placentas demonstrated significant alterations of transcripts coding for a variety of molecules involved in blood pressure regulation, inflammation, and apoptosis [74]. Moreover, loss of p57 seems to be associated with placentomegaly due to unrestricted endoreduplication [75]. The role of p57 in placental development needs further investigation.
It has been shown that the co-expression of p53 and p21 can lead to cell cycle arrest via suppression of G2 phase and mitotic (M) genes by the DREAM (Dimerization partner, RB-like proteins, E2Fs And Multi-vulval B) multiprotein complex [76,77]. In cancer cells, loss of p21 reduced the DREAM binding to the cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) of the promoters, resulting in the expression of G2/M genes [77]. However, microarray analysis using BeWo cells depleted of p21 did not show an increase in G2/M genes, which could be attributed to their long cell doubling time [78]. Remarkably, it revealed other interesting candidate genes such as the elevated gene TXNIP, which encodes the protein thioredoxin interacting protein, a major regulator of the cellular redox signaling, protecting cells from oxidative stress [79]. Interestingly, TXNIP was expressed in the STB, CTBs, and endothelial cells of the human placenta, where its expression was increased in the second and third trimester [80]. TXNIP loss significantly increased p21 protein expression levels in ARPE-19 cells, inhibiting cell growth [81], whereas TXNIP stabilized p27 protein indirectly in fibroblasts [82]. Further investigations are required to study the relationship of p21 with these candidate genes and their functions in PE.

Conclusions
Taken together, we show that p21 and its family members p27 and p57 are highly expressed in the first trimester of pregnancy, pointing to their importance in early placental development. The expression of p21 in CTBs and the STB is further highlighted with 3D trophoblast organoids, which have been reported to mimic the placental villi structurally, phenotypically, metabolically, and endocrinologically [28,29]. Moreover, we report that p21 is reduced in early-onset PE fCTBs ongoing to fuse to the STB. This finding is further underscored by our observation that hypoxia reduces p21 in trophoblastic cells and organoids. Depletion of p21 decreases the expression of fusion-related genes such as syncytin-2 and impairs the fusion capability of trophoblasts, characteristic of PE. Moreover, the percentages of positive p57 fCTBs and EVTs are significantly reduced in early-onset PE placentas, indicating its potential involvement in CTB fusion and EVT migration. These data strongly suggest that p21 deficiency, in collaboration with reduced p57, is likely to contribute to the pathogenesis of early-onset PE.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cells10092214/s1, Figure S1: Characterization of long-term trophoblast organoid culture, Figure S2: Expression of cell cycle regulators in late-onset PE, Figure S3: Characterization of primary cytotrophoblasts. Figure S4: Raw data of all western blots. Table S1: Data from whole gene-expression array with a p-value smaller than 0.05.