TY - JOUR
AU1 - Wang,, Hao
AU2 - Xu,, Ping
AU3 - Luo,, Xiaofang
AU4 - Hu,, Mingyu
AU5 - Liu,, Yamin
AU6 - Yang,, Yike
AU7 - Peng,, Wei
AU8 - Bai,, Yuxiang
AU9 - Chen,, Xuehai
AU1 - Tan,, Bin
AU1 - Wu,, Yue
AU1 - Wen,, Li
AU1 - Gao,, Rufei
AU1 - Tong,, Chao
AU1 - Qi,, Hongbo
AU1 - Kilby, Mark, D
AU1 - Saffery,, Richard
AU1 - Baker, Philip, N
AB - Abstract Fetal growth restriction (FGR) is a condition in which a newborn fails to achieve his or her prospective hereditary growth potential. This condition is associated with high newborn mortality, second only to that associated with premature birth. FGR is associated with maternal, fetal, and placental abnormalities. Although the placenta is considered to be an important organ for supplying nutrition for fetal growth, research on FGR is limited, and treatment through the placenta remains challenging, as neither proper uterine intervention nor its pathogenesis have been fully elucidated. Yes-associated protein (YAP), as the effector of the Hippo pathway, is widely known to regulate organ growth and cancer development. Therefore, the correlation of the placenta and YAP was investigated to elucidate the pathogenic mechanism of FGR. Placental samples from humans and mice were collected for histological and biomechanical analysis. After investigating the location and role of YAP in the placenta by immunohistochemistry, we observed that YAP and cytokeratin 7 have corresponding locations in human and mouse placentas. Moreover, phosphorylated YAP (p-YAP) was upregulated in FGR and gradually increased as gestational age increased during pregnancy. Cell function experiments and mRNA-Seq demonstrated impaired YAP activity mediated by extracellular signal-regulated kinase inhibition. Established FGR-like mice also recapitulated a number of the features of human FGR. The results of this study may help to elucidate the association of FGR development with YAP and provide an intrauterine target that may be helpful in alleviating placental dysfunction. Introduction Fetal growth restriction (FGR), also known as intrauterine growth restriction (IUGR), is a condition in which a newborn fails to reach developmental potential and exhibits fetal weight below the 10th percentile for gestational age [1–3]. FGR is a widely considered to be a serious complication affecting up to 5–10% of pregnancies globally [4]. This condition is associated with several adverse short-term or long-term health problems in newborns, such as type 2 diabetes, neonatal hypoglycemia, obesity, hypertension, and coronary heart disease [5–7]. Although the etiology of FGR is complex and not completely elucidated, the development of FGR can be attributed to placental, maternal, and fetal factors [8, 9], while impaired placental function is considered to be the most critical cause of FGR because of the vital role of the placenta in providing essential conditions for fetal growth in utero [10, 11]. Accumulating evidence has shown that excessive apoptosis, autophagy, oxidative damage, deficient remodeling of the uterine spiral artery, and deregulation of angiogenesis are highly correlated with the placenta of FGR [12–15]. The process by which trophoblast cells invade the maternal uterine tissue is a specific and sustaining biological behavior known as placentation, where appropriate remodeling of the maternal vasculature primarily relies on the migration and invasion of trophoblast cells [16]. During pregnancy, the capabilities of invasion and migration of trophoblast cells are crucial in various physiological processes associated with placental and fetal development [17]. Inappropriate invasion or migration of trophoblasts causes placental insufficiency, which leads to ischemia, hypoxia, or hypoalimentation of the fetus, which are considered causes of FGR. These defects ultimately affect organogenesis and physical and neural development [11, 18]. The maternal–fetal interface is crucial for the exchange of various growth factors, immunoproteins, and hormones between the fetus and mother [19, 20]. The process of placental dysfunction leading to FGR is inconspicuous, and the poor outcome is generally discovered at birth, when the newborn fails to meet the expected growth profile or during Doppler examination when the fetus is far behind in body proportions compared with other fetuses at the same period [21, 22]. Procedures for an FGR delivery are usually confined to fetal monitoring and proper management for preterm delivery if indicated. Unfortunately, there is no optimal treatment to prevent or improve placental insufficiency, FGR, and its short- and long-term complications. The Hippo pathway was discovered to be highly conserved in regulating organ size in mammalian cells, especially in cancers [23, 24]. Mst1/2 and Lats1/2 are known upstream components of the Hippo pathway, and their effectors are Yes-associated protein (YAP)/PDZ-binding motif (TAZ) coactivators [25]. YAP is phosphorylated upstream under unfavorable conditions (energy shortage, hypoxia, or contact inhibition), leading to a decrease in transcriptional activity [26, 27]. In recent years, the function and regulation of YAP have been well studied in cancers, while Hippo/YAP in the reproductive area has not been sufficiently explored. Previous studies revealed that decidualization is critical for the initiation of pregnancy mediated by the Hippo signaling pathway, which plays a significant role in proliferation processes. Furthermore, YAP in decidual cells from pregnant women was expressed considerably more strongly than in decidual cells from nonpregnant women, indicating an essential but unclear role in pregnancy [28, 29]. In mouse embryo development, Hippo signaling is crucial to the formation of two cell lineages at an early stage of life: the trophectoderm (which gives rise to the placenta) and inner cell mass (which gives rise to the embryo proper and yolk sac) [30, 31]. Other factors that contribute to FGR include aberrant expression of specific genes and adverse maternal environment. One factor to consider is the Hippo signaling pathway, which has a crucial role in organ growth control during development, and its dysregulation contributes to tissue and organ overgrowth [32, 33]. Trophoblast cells of the placenta have similar characteristics to cancer cells [34]; however, the role of YAP in placental development has not been elucidated. In this study, we identified the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway as a putative upstream branch of YAP in trophoblast cells. In HTR-8/SVneo and JAR cell lines, downregulation of MAPK/ERK upregulates p-YAP expression, thereby promoting Hippo signaling activity and weakening Hippo target gene expression. On the other hand, overexpression of YAP regulates Hippo signaling in trophoblasts and rescues part of the MAPK-ERK adverse impact on trophoblast function. To date, there have been few studies on YAP function during pregnancy or pregnancy complications, and the association of FGR with YAP is unknown. The present study aimed to reveal the involvement of the Hippo pathway in the pathogenesis of FGR. Materials and methods Ethics statement The study design involving patients and animals was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (No: 2018-109) and in accordance with the principles set out in the Declaration of Helsinki. All clinical samples were collected with written informed consent provided by the participant patients. The animals were regularly checked by qualified administrators for health monitoring, environment supervision, animal welfare assurance, and operation revision. All subsequent surgeries on animals were performed cautiously under normative anesthesia to minimize their suffering. Patient recruitment and sample collection Placental tissues at the maternal face from normal pregnancies (n = 30) and FGR patients (n = 28) who were admitted to the First Affiliated Hospital of Chongqing Medical University (No: 2018-109) for cesarean deliveries were collected as previously established in our group [35–37]. FGR was defined as newborns having a birthweight below the 10th percentile [38]. Patients with other major pregnancy complications, such as gestational diabetes mellitus, premature rupture of membranes, renal disease, premature labor, or preeclampsia, were excluded. The patients’ clinical features are characterized in Table S1. Placenta villous tissue of the first trimester (8 weeks of gestational age, n = 4) and placentas from the second trimester (14, 17, and 21 weeks of gestational age, n = 3, respectively) were collected from women who underwent legal termination due not to medical reasons, history of spontaneous abortion or ectopic pregnancy. For these tissues, a portion was fixed in 4% paraformaldehyde and then embedded in paraffin for further immunohistochemistry (IHC) analysis. A portion was dehydrated and then transferred to O.C.T. compound for further immunofluorescence (IF) analysis. A portion was stored at −80 °C for Western blotting. A portion was kept in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) for further qRT-PCR analysis. The rest was immediately snap frozen in liquid nitrogen and stored at −80 °C for preservation. Procedures of intraperitoneal injection of SCH772984 (SCH) Eight- to 12-week-old C57BL/6 J female mice weighing 18–22 g ordered from the Experimental Animal Center of Chongqing Medical University were randomly mated with age-matched male mice. Mice were considered to be in embryonic stage 0.5 (E0.5) with the observation of a vaginal plug after the day of mating. All mice were kept in a temperature-controlled room (23 °C) with a 12:12 h light–dark cycle. Pregnant mice for modeling were randomly assigned into three groups (Con, n = 6; Vehicle, n = 6; SCH, n = 6). On E8.5, the pregnant mice were intraperitoneally injected with vehicle mixture (5% DMSO + 30% polyethylene glycol 300 + ddH2O) or SCH772984 (20 mg/kg, Selleckchem, Houston, TX, USA) daily, and the Con group was designed as no treatment throughout gestation [39, 40]. The mice were euthanized on E18.5 for further analysis: morphology recording, sample collection, and related measurements. Sample collection was in accordance with clinical samples. Cell culture The immortalized human trophoblast cell line HTR-8/SVneo was purchased from American Type Culture Collection (Manassas, VA, USA). The human choriocarcinoma cell line JAR was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA), which was supplemented with 10% fetal bovine serum (FBS) (GIBCO, Invitrogen, New York, USA) and 1% penicillin–streptomycin at 37 °C under 5% CO2 humidified air. Inhibition of YAP nuclear export RPMI 1640 medium (no glucose) (Thermo Fisher Scientific, Waltham, MA, USA) and supplement without FBS were used for stress-induced YAP cytoplasm localization attributed to glucose starvation or serum deprivation [41, 42]. When grown to 70–80% confluence, YAP export from the nucleus was induced by glucose starvation or serum deprivation for 24 h. Leptomycin B (25 nM, LMB, Beyotime Institute of Biotechnology, Shanghai, China) was added 30 min ahead of harvest at each time point to inhibit nuclear export of YAP. Lentivirus infection To overexpress YAP in HTR-8/SVneo and JAR cells, cells were infected with lentivirus carrying the YAP gene (GeneChem, Montreal, QC, Canada). The retroviral vector GV358 was constructed in detail, and sequences for the YAP overexpression gene are shown in Figure S1a. A total of 1 × 105 HTR-8/SVneo and JAR cells were transfected with 2 × 106 virus (multiplicity of infection = 20) for 48 h in the presence of polybrene according to the manufacturer’s instructions. Then, the cells were transferred to fresh medium containing puromycin (2 μg/ml) for the selection of stable clones after three passages. The sequences for the YAP-overexpression gene are listed in Figure S1b. Immunohistochemistry The human placental tissues were washed with PBS and fixed in 4% paraformaldehyde at room temperature overnight. Then, the samples were dehydrated and embedded in paraffin before sectioning into 4-mm-thick sections. The sections were deparaffinized at 65 °C for 2 h, immersed in xylene for 30 min twice and rehydrated in a serial ethanol gradient (100–95–85–75%). The samples were heated in a microwave in citric sodium (10 mM, pH 6.0) for 15 min to retrieve antigens. Cells were blocked with 3% peroxide–methanol at room temperature for 10 min for endogenous peroxidase ablation. After that step, sections were incubated with rabbit primary mAb against YAP (1:100; Abcam, Cambridge, UK) and mouse mAb against cytokeratin 7 (CK7) (1:100; Abcam, Cambridge, UK) at 4 °C overnight. The next day, secondary antibody conjugated with horseradish peroxidase (Solarbio Life Sciences, Beijing, China) was applied for 1 h at room temperature followed by diaminobenzidine solution development. The images were captured by an Evos Fl Color Imaging System (Thermo Fisher Scientific, USA). Immunofluorescence Cells were fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and then incubated with rabbit primary mAb against YAP (1:100; Abcam, Cambridge, UK), mouse mAb against CK7 (1:100; Abcam, Cambridge, UK), or rabbit primary mAb against p-YAP (1:100; Affinity Biosciences, Cincinnati, OH, USA) overnight at 4 °C, followed by incubation with FITC-conjugated or cyanine 3-conjugated goat anti-rabbit (1:100; Proteintech Group, Inc., Rosement, IL 60018, USA) for 1 h at room temperature. For human and mouse placenta staining, frozen sections were treated with the indicated antibodies. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and images were visualized using an Evos Fl Color Imaging System (Thermo Fisher Scientific, USA). Western blotting Protein extracts were prepared from placental tissues and cells using RIPA lysis buffer supplemented with 1% cocktail phosphatase inhibitor and 1% cocktail protease inhibitor (Bimake, Houston, TX, USA). The lysates were then separated by SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Sigma, USA). After blocking with 5% nonfat dry milk (Bio-Rad) in Tris buffer containing |$5\textperthousand$| Tween-20 for 1 h at room temperature, the membranes were immunoblotted with primary antibodies against YAP (1:200; Santa), p-YAPSer127 (1:5000; Abcam), TATA-binding protein (TBP) (1:1000; CST), or β-actin (1:1000; Proteintech Group, Inc., Rosement, IL 60018, USA) overnight at 4 °C. After rinsing with Tris buffer containing |$5\textperthousand$| Tween-20, the membranes were then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000; ZSGB-BIO, Beijing, China) at room temperature for 1 h. Immunoreactive bands were developed using an enhanced chemiluminescent substrate (Millipore Sigma, USA), and the images were captured and analyzed by a ChemiDoc XRS+ system (Bio-Rad, Hercules, California, USA). Extraction of nuclear and cytoplasmic proteins Cellular cytoplasmic and nuclear extracts were harvested and washed with cold PBS, and then the buffer was aspirated completely. Next, 200 μl cytoplasmic extraction buffer was added (Invent Biotechnologies, Beijing, China), swirling was employed to distribute the lysis buffer over the entire surface of the tissue culture, and the tissue culture was placed on ice for 5 min. The lysed cells were scraped with a pipette tip, and the cell lysate was transferred to a prechilled 1.5-ml microcentrifuge tube. The tube was vortexed vigorously for 15 s. Then, the tube was centrifuged for 5 min at top speed in a microcentrifuge at 4 °C. The supernatant (cytosol fraction) was transferred to a fresh prechilled 1.5-ml tube to obtain the cytoplasmic protein. Appropriate amounts of nuclear extraction buffer were added to the pellet, which was vortexed vigorously for 15 s, and the tube was incubated on ice for 1 min. The 15-s vortexing step and 1-min incubation step were repeated four times. Finally, the nuclear extract was transferred to a prechilled filter cartridge with a collection tube and centrifuged at top speed in a microcentrifuge for 30 s. The filter cartridge was discarded. Nuclear extract was stored at −80 °C for subsequent Western blotting. Matrigel invasion assay The invasion assay was performed in a Transwell chamber consisting of a 24-well plate with membrane inserts (Corning, New York, NY, USA) containing polycarbonate filters with 8-mm pore sizes precoated with 60 μl of 1 mg/ml matrigel matrix solution (BD Biosciences, USA). A suspension of 4 × 104 cells in 200 μl serum-free culture medium was added to the inserts, and each insert was placed in the lower chamber containing 600 μl of 10% FBS culture medium. After 24 h, the cells that penetrated the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. The cell migration assay was similar to the invasion assay, but the insert was not coated with matrigel. Images were captured by using the Evos Fl Color Imaging System. Wound-healing assay Subconfluent cultures of HTR-8/SVneo and JAR cell lines were applied. The plates were scratched by a 200-μl pipette tip after the addition of SCH772984 (ERK inhibitor, ERKi) for 48 h. Phase contrast images were taken with an Evos Fl Color Imaging System (Thermo Fisher Scientific) at the time of the scratch (0 h) and 48 h after. Scratch areas were quantified using ImageJ software. Wound closure rates were calculated as percentages of the initial area (0 h) and normalized with the control group. RNA extraction and qRT-PCR Total RNA was extracted from HTR-8/SVneo and JAR cell lines, mouse and human placenta using RNAiso Plus (Takara, Dalian, China), and the RNA concentration was measured by ultraviolet spectroscopy (Nanodrop 2000; Thermo, Massachusetts, USA). Total RNA (1 μg) was used for reverse transcription with a Prime Script RT reagent kit (Roche Life Science, Germany). Primers were designed and synthesized by TaKaRa (Dalian, China); β-actin was used as an endogenous control for gene expression analysis. The sequences of the PCR primer pairs for each gene are shown in Table S2. PCR cycling conditions included predenaturing at 95 °C for 3 min followed by 40 cycles (94 °C for 5 s, 58 °C for 15 s, and 72 °C for 15 s) and extension at 72 °C for 30 s. The mean threshold cycle (Ct) values were normalized to β-actin, and the relative mRNA levels of CTGF, CYR61, and AMOTL2 were analyzed. Alizarin red/Alcian blue staining of E18.5 embryo skeletons The Alizarin red/Alcian blue double staining technique was modified from Whitaker and Dix [43]. Briefly, E18.5 pups were fixed in 10% buffered formalin overnight followed by careful skin and viscera removal. The pups were fixed in 95% ethanol overnight, then lipids were removed in acetone overnight, and the pups were immersed in staining solution (0.03% Alcian blue 8GX, 0.006% Alizarin red, and 10% acetic acid dissolved in 95% ethanol) for 72 h. Soft tissues were cleared in 1% KOH until bone (red) and cartilage (blue) could be distinctly observed. Finally, the well-stained pups were stored in daily increasing ratios of glycerin/potassium hydroxide (KOH) to pure glycerin. All of the above operations were performed on a slow rocker (45–55 rpm) at room temperature. Statistical analysis Data are presented as the mean ± SEM. The significance of the results was assessed by a Mann–Whitney U test or Student t-test using the GraphPad Prism software package (version 7.0; La Jolla, California, USA), with a P value less than 0.05 being considered to be significant. Figure 1 Open in new tabDownload slide Phosphorylation of YAP is upregulated in the trophoblasts of FGR-complicated placentas. (a) IHC staining of YAP and CK7 in first trimester villi of human placenta. CK7 was regarded as a positive control of STB and CTB in trophoblasts; scale bars, 100 μm. (b) IHC staining of YAP and CK7 in normal and FGR term placenta. CK7 was regarded as a positive control of STB and CTB in trophoblasts; scale bar, 100 μm. (c) IHC staining of YAP and CK7 in E18.5 C57BL/6 J mouse placenta. CK7 was regarded as a positive control of trophoblasts. (d) Western blotting of YAP and p-YAP in normal and FGR placentas. **P < 0.01; Mann–Whitney U test. (e) mRNA levels of CTGF, CYR61, and AMOTL2 were measured by qRT-PCR and normalized to β-actin. **P < 0.01, *P < 0.05; Mann–Whitney U test. All data are presented as the mean ± SEM. Figure 1 Open in new tabDownload slide Phosphorylation of YAP is upregulated in the trophoblasts of FGR-complicated placentas. (a) IHC staining of YAP and CK7 in first trimester villi of human placenta. CK7 was regarded as a positive control of STB and CTB in trophoblasts; scale bars, 100 μm. (b) IHC staining of YAP and CK7 in normal and FGR term placenta. CK7 was regarded as a positive control of STB and CTB in trophoblasts; scale bar, 100 μm. (c) IHC staining of YAP and CK7 in E18.5 C57BL/6 J mouse placenta. CK7 was regarded as a positive control of trophoblasts. (d) Western blotting of YAP and p-YAP in normal and FGR placentas. **P < 0.01; Mann–Whitney U test. (e) mRNA levels of CTGF, CYR61, and AMOTL2 were measured by qRT-PCR and normalized to β-actin. **P < 0.01, *P < 0.05; Mann–Whitney U test. All data are presented as the mean ± SEM. Results Phosphorylation of YAP is upregulated in the trophoblasts of FGR complicated placenta FGR has long been attributed to inadequate placental development, and YAP plays an important role in the regulation of organ growth during development. Therefore, the protein expression patterns of YAP in first trimester villi, human term placenta, and E18.5 mouse placenta were assessed by IHC with serial sections, which showed that in human, YAP was largely co-stained with CK7 in cytotrophoblasts (CTBs) and syncytrophoblasts (STBs) (Figure 1a and b). In mouse, YAP was abundant in the labyrinth zone (Lz) but relatively low in junctional zone (Jz) and decidual tissue (De). Moreover, the total protein expression levels of YAP did not differ between FGR and normal placenta (Figure 1c). However, the phosphorylation level of YAP was significantly higher in FGR placentas than in normal placentas (Figure 1d). It is known that unphosphorylated YAP translocates into the nucleus to execute the function of a transcriptional coactivator and thus regulates the expression of genes involved in proliferation and angiogenesis [44]. To ascertain whether aberrant YAP phosphorylation leads to changes in the transcription of its downstream genes, the mRNA levels of CTGF, CYR61, and AMOTL2 were examined by qRT-PCR, and all of these genes were significantly downregulated in FGR placenta (Figure 1e), implying that the translocation of YAP into the nucleus might be constrained in FGR-complicated placenta. Phosphorylation of YAP in the placenta ascends throughout pregnancy To characterize the expression and phosphorylation patterns of placental YAP during pregnancy, human placentas collected at various gestational ages were subjected to assessment of total YAP and p-YAP. IF staining demonstrated that p-YAP expression was remarkably increased with gestational age in the human placenta, while the total expression of YAP protein was not, and both of these findings were further confirmed by Western blotting (Figure 2a and b). To ascertain whether the mouse placenta has similar patterns, the levels of YAP and p-YAP in the mouse placenta throughout pregnancy were determined. In line with the human placenta, the phosphorylation level of YAP was quite low between E8.5 and E12.5 but increased immediately after placentation on E13.5 and then further increased as gestational age was prolonged, while total YAP protein levels remained stable during pregnancy (Figure 2c and d). Given that unphosphorylated YAP enters the nucleus to enhance the expression of genes involved in organ growth, and our results elucidate the association between FGR and excessive YAP phosphorylation in placenta, we assume that phosphorylation of YAP in trophoblasts might be a “brake” for the development of the placenta. Figure 2 Open in new tabDownload slide Phosphorylation of YAP in the placenta ascends throughout pregnancy. (a) Images of YAP and p-YAP IF staining of gradual gestational age (week-9, week-14, week-17, week-21, and week-40) in human placenta. Scale bars, 200 μm. (b) Expression of p-YAP and YAP in human placenta with progressive gestational age determined by Western blotting. (c) Scanning images of YAP and p-YAP IF staining of gradual gestational age (E8.5, E9.5, E10.5, E12.5, E13.5, E15.5, E16.5, and E18.5) in mouse placenta. (d) Expression of p-YAP and YAP in mouse placenta with increasing gestational age determined by Western blotting. Figure 2 Open in new tabDownload slide Phosphorylation of YAP in the placenta ascends throughout pregnancy. (a) Images of YAP and p-YAP IF staining of gradual gestational age (week-9, week-14, week-17, week-21, and week-40) in human placenta. Scale bars, 200 μm. (b) Expression of p-YAP and YAP in human placenta with progressive gestational age determined by Western blotting. (c) Scanning images of YAP and p-YAP IF staining of gradual gestational age (E8.5, E9.5, E10.5, E12.5, E13.5, E15.5, E16.5, and E18.5) in mouse placenta. (d) Expression of p-YAP and YAP in mouse placenta with increasing gestational age determined by Western blotting. Figure 3 Open in new tabDownload slide Analysis of YAP expression after ERK inhibitor treatment. (a) ERK phosphorylation in HTR-8/SVneo cells in response to various doses (10 nM to 10 μM) of SCH772984 as determined by Western blotting. (b) The phosphorylation of ERK and YAP in HTR-8/SVneo cells in response to SCH772984 (100 nM) treatment over a 72-h time course. (c) The effects of SCH772984 (100 nM) treatment on HTR-8/SVneo cell proliferation over a time course determined by EdU (red) staining. The nuclei were counterstained with DAPI (blue). Scale bars, 400 μm. ***P < 0.001; one-way ANOVA. (d) CCK-8 assay on HTR-8/SVneo cells at 0, 24, 48, and 72 h after SCH772984 (100 nM) treatment. OD, optical density. **P < 0.01; one-way ANOVA. (e) Cell cycle analysis of HTR-8/SVneo cells by flow cytometry at 0, 24, 48, and 72 h after SCH772984 (100 nM) treatment. **P < 0.01, ***P < 0.001, NS indicates nonsignificant; one-way ANOVA. All data are presented as the mean ± SEM. Figure 3 Open in new tabDownload slide Analysis of YAP expression after ERK inhibitor treatment. (a) ERK phosphorylation in HTR-8/SVneo cells in response to various doses (10 nM to 10 μM) of SCH772984 as determined by Western blotting. (b) The phosphorylation of ERK and YAP in HTR-8/SVneo cells in response to SCH772984 (100 nM) treatment over a 72-h time course. (c) The effects of SCH772984 (100 nM) treatment on HTR-8/SVneo cell proliferation over a time course determined by EdU (red) staining. The nuclei were counterstained with DAPI (blue). Scale bars, 400 μm. ***P < 0.001; one-way ANOVA. (d) CCK-8 assay on HTR-8/SVneo cells at 0, 24, 48, and 72 h after SCH772984 (100 nM) treatment. OD, optical density. **P < 0.01; one-way ANOVA. (e) Cell cycle analysis of HTR-8/SVneo cells by flow cytometry at 0, 24, 48, and 72 h after SCH772984 (100 nM) treatment. **P < 0.01, ***P < 0.001, NS indicates nonsignificant; one-way ANOVA. All data are presented as the mean ± SEM. Analysis of YAP expression after ERK inhibitor treatment ERK is an evolutionarily conserved component of MAPK signaling, which is mediated by a wide range of extracellular stimuli to regulate proliferation, survival, differentiation, and migration [45, 46]. Previous reports have shown that the MAPK/p38 or MAPK/Jnk pathway regulates YAP activity and the expression of Hippo downstream targets in mammalian cells [47, 48], but whether the MAPK/ERK pathway has regulatory effects on YAP in trophoblasts has not been determined. Thus, we analyzed the expression levels of YAP and p-YAP after ERK inhibition. First, various doses of the ERK-specific inhibitor SCH772984 were applied to HTR-8/SVneo trophoblast cells, and dosages greater than 100 nM significantly suppressed ERK phosphorylation (Figure 3a). We then examined the impact of ERK inhibition on the Hippo pathway and found that after treatment with 100 nM SCH772984 for 24, 48 and 72 h, although ERK phosphorylation was efficiently suppressed within 24 h, the phosphorylation of YAP was upregulated after 48 h (Figure 3b). 5-Ethynyl-2′-deoxyuridine (EdU) staining showed that DNA synthesis in HTR-8/SVneo cells was significantly repressed after 72 h of SCH772984 treatment (Figure 3c). Furthermore, the results of cell counting kit-8 (CCK8) assays demonstrated that trophoblast proliferation was notably decreased after 72 h of SCH772984 treatment (Figure 3d). In line with these data, flow cytometry revealed that cell cycle arrest in G1 phase was induced at 72 h after the inhibition of ERK (Figure 3e). The relative long-term interval between p-ERK inhibition and YAP phosphorylation or between YAP phosphorylation and downregulation of cell viability not only confirmed the order of these events in trophoblasts but also implied that these events are likely induced by intermediate gene expression, rather than direct phosphorylation via signal transduction. Figure 4 Open in new tabDownload slide Upregulated YAP rescued invasion and migration ability impaired by ERK inhibition in trophoblast cell lines. Stable YAP-overexpressing HTR-8/SVneo (a) and JAR (c) cells were established by lentivirus infection (Ubi-MCS-EGFP). Phase contrast, PC. Confirmation of YAP overexpression in HTR-8/SVneo (b) and JAR (d) cells by Western blot. (e) Cell migration ability in HTR-8/SVneo cells after SCH772984 (100 nM) treatment for 24 h and quantification (f). Scale bar, 400 μm. **P < 0.01, ****P < 0.0001; two-way ANOVA. Cell invasion ability in HTR-8/SVneo (g, h) and JAR (i, j) cells after SCH772984 (100 nM) treatment for 24 h. *P < 0.05, **P < 0.01, ****P < 0.0001; two-way ANOVA. All data are presented as the mean ± SEM. Figure 4 Open in new tabDownload slide Upregulated YAP rescued invasion and migration ability impaired by ERK inhibition in trophoblast cell lines. Stable YAP-overexpressing HTR-8/SVneo (a) and JAR (c) cells were established by lentivirus infection (Ubi-MCS-EGFP). Phase contrast, PC. Confirmation of YAP overexpression in HTR-8/SVneo (b) and JAR (d) cells by Western blot. (e) Cell migration ability in HTR-8/SVneo cells after SCH772984 (100 nM) treatment for 24 h and quantification (f). Scale bar, 400 μm. **P < 0.01, ****P < 0.0001; two-way ANOVA. Cell invasion ability in HTR-8/SVneo (g, h) and JAR (i, j) cells after SCH772984 (100 nM) treatment for 24 h. *P < 0.05, **P < 0.01, ****P < 0.0001; two-way ANOVA. All data are presented as the mean ± SEM. Upregulated YAP rescued invasion and migration ability impaired by ERK inhibition in trophoblast cell lines To study the role of the ERK-YAP signaling pathway in trophoblastic functions, GFP-tagged YAP was overexpressed (YAP-OE) in HTR-8/SVneo and JAR cells (Figure 4a–d). Then, these cells were subjected to invasion and migration assessments along with wild-type controls. Matrigel-based transwell assay results showed that the invasion of HTR-8/SVneo cells was significantly inhibited by the presence of SCH772984 but promoted by overexpression of YAP. Accordingly, the inhibitory effects of SCH772984 on invasiveness were largely blocked in YAP-OE HTR-8/SVneo cells (Figure 4e and f). Similarly, SCH772984 treatment significantly inhibited migration in both cell lines, but this impairment was largely rescued in YAP-OE HTR-8/SVneo and JAR cells (Figure 4g–j). Figure 5 Open in new tabDownload slide ERK inhibition induces YAP phosphorylation and inactivation but is partly reversed by YAP-OE. Endogenous YAP localization by IF of HTR-8/SVneo (a) and JAR (d) cells. Nuclei were visualized with DAPI staining. Scale bars = 20 μm. Western blotting of the subcellular localization of YAP in HTR-8/SVneo (b) and JAR (e) cells. β-actin was regarded as the cytoplasmic control, and TBP was regarded as the nuclear control. The mRNA levels of CTGF, CYR61, and AMOTL2 in HTR-8/SVneo (c) and JAR (f) cells were measured by qRT-PCR and normalized to the β-actin control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA. All data are presented as the mean ± SEM. Figure 5 Open in new tabDownload slide ERK inhibition induces YAP phosphorylation and inactivation but is partly reversed by YAP-OE. Endogenous YAP localization by IF of HTR-8/SVneo (a) and JAR (d) cells. Nuclei were visualized with DAPI staining. Scale bars = 20 μm. Western blotting of the subcellular localization of YAP in HTR-8/SVneo (b) and JAR (e) cells. β-actin was regarded as the cytoplasmic control, and TBP was regarded as the nuclear control. The mRNA levels of CTGF, CYR61, and AMOTL2 in HTR-8/SVneo (c) and JAR (f) cells were measured by qRT-PCR and normalized to the β-actin control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA. All data are presented as the mean ± SEM. ERK inhibition induces YAP phosphorylation and inactivation but is partly reversed by YAP-OE To investigate the potential role of ERK on the regulation of subcellular distribution of YAP in trophoblasts. The localization of YAP in HTR-8/SVneo and JAR cells was determined by IF staining. As shown in Figure 5a and d, YAP is predominantly localized in the nucleus of trophoblasts, and the inhibition of ERK by SCH772984 (100 nM) for 24 h resulted in increased cytoplasmic YAP; however, this loss of nuclear YAP can be partially rescued by overexpression of YAP. Western blotting of the above groups was applied to confirm the observed results in HTR-8/SVneo and JAR cells (Figure 5b and e). To further validate the transcript level of downstream targets induced by endonuclear YAP, qRT-PCR for CTGF, CYR61, and AMOTL2 was applied. As a result, these genes were significantly upregulated in the YAP-OE group and inhibited after ERK inhibitor treatment but partly reversed by YAP-OE (Figure 5c and f). To study whether the subcellular localization of YAP corresponds to its phosphorylation status, YAP phosphorylation in HTR-8/SVneo cells was induced by serum deprivation or glucose starvation, both of which enhance YAP phosphorylation in a time-dependent manner; nevertheless, treatment with leptomycin B (LMB), a nuclear transport inhibitor, largely suppressed serum deprivation- or glucose starvation-induced YAP phosphorylation (Figure S2). Defective development and p-YAP elevation are associated with the placenta of FGR mice To reveal whether ERK deficiency is a causative factor for FGR, ERK-deficient mice were generated by daily intraperitoneal injection of ERK inhibitor from E8.5 to E17.5. Unlike ERK knockout mice exhibiting serious morphological defects and skeletal retardation [49], our ERK inhibition treatment-induced FGR-like mice showed smaller placental and fetal sizes without skeletal defects or delays (Figure 6a–c). Our data showed that the phosphorylation of ERK was strikingly downregulated in the placenta from ERK-deficient mice on E18.5, while the phosphorylation of YAP was correspondingly elevated compared to the control group (Figure 6d). Moreover, YAP in the nucleus was considerably lower than that in the vehicle group, while the p-YAP level was considerably higher in the cytoplasm, which was in keeping with the in vitro findings, and this translocation of YAP was visualized by IF staining and validated by Western blotting (Figure 6e and f). Accordingly, the expression levels of CTGF, CYR61, and AMOTL2 in the placenta were all significantly downregulated by ERK inhibition (Figure 6g). Histological analyses of the uteroplacental unit by H&E staining on E18.5 revealed a significant reduction in the labyrinth area in ERK-inhibited mice, while the junctional zone area was augmented (Figure 6h). The reduction of positive staining of Ki67 in ERK-inhibited placenta presented an impaired proliferation ability in the labyrinth area (Figure 6i). Figure 6 Open in new tabDownload slide Defective development and p-YAP elevation are associated with the placenta of FGR mice. (a) Phenotypic appearance of ERK inhibition (SCH) fetuses and placenta at E18.5. The SCH group exhibited a smaller body and placenta size. Scale bar, 10 mm. (b) Pup weight, crown-rump length, and placental weight on E18.5. **P < 0.01, ***P < 0.001, NS indicates nonsignificant; one-way ANOVA. (c) Alizarin red/Alcian blue staining of E18.5 fetuses. Scale bar, 10 mm. (d) Western blotting of p-ERK, ERK, p-YAP, and YAP in placenta collected from the blank, vehicle, and SCH groups at E18.5. ***P < 0.001, ****P < 0.0001, NS indicates nonsignificant; one-way ANOVA. (e) YAP staining by IF of placenta from vehicle and SCH groups at E18.5. Scale bars, 100 μm. Insets: higher magnification views of the boxed areas. (f) Confirmation of subcellular localization of YAP in placenta from the vehicle and SCH groups at E18.5 by Western blotting. *P < 0.05, **P < 0.01; Mann–Whitney U test. (g) mRNA levels of CTGF, CYR61, and AMOTL2 were measured in placenta from the blank, vehicle, and SCH groups at E18.5 by qRT-PCR and normalized to the β-actin control. **P < 0.01, ***P < 0.001; one-way ANOVA. (h) Hematoxylin and eosin (H&E) staining of placenta from the vehicle and SCH groups at E18.5. The areas of Lz and Jz were analyzed. Scale bars, 400 μm. *P < 0.05; Mann–Whitney U test. (i) IHC staining of Ki67 in placenta from the vehicle and SCH groups at E18.5. Scale bars, 200 μm. **P < 0.01; Mann–Whitney U test. All data are presented as the mean ± SEM. Figure 6 Open in new tabDownload slide Defective development and p-YAP elevation are associated with the placenta of FGR mice. (a) Phenotypic appearance of ERK inhibition (SCH) fetuses and placenta at E18.5. The SCH group exhibited a smaller body and placenta size. Scale bar, 10 mm. (b) Pup weight, crown-rump length, and placental weight on E18.5. **P < 0.01, ***P < 0.001, NS indicates nonsignificant; one-way ANOVA. (c) Alizarin red/Alcian blue staining of E18.5 fetuses. Scale bar, 10 mm. (d) Western blotting of p-ERK, ERK, p-YAP, and YAP in placenta collected from the blank, vehicle, and SCH groups at E18.5. ***P < 0.001, ****P < 0.0001, NS indicates nonsignificant; one-way ANOVA. (e) YAP staining by IF of placenta from vehicle and SCH groups at E18.5. Scale bars, 100 μm. Insets: higher magnification views of the boxed areas. (f) Confirmation of subcellular localization of YAP in placenta from the vehicle and SCH groups at E18.5 by Western blotting. *P < 0.05, **P < 0.01; Mann–Whitney U test. (g) mRNA levels of CTGF, CYR61, and AMOTL2 were measured in placenta from the blank, vehicle, and SCH groups at E18.5 by qRT-PCR and normalized to the β-actin control. **P < 0.01, ***P < 0.001; one-way ANOVA. (h) Hematoxylin and eosin (H&E) staining of placenta from the vehicle and SCH groups at E18.5. The areas of Lz and Jz were analyzed. Scale bars, 400 μm. *P < 0.05; Mann–Whitney U test. (i) IHC staining of Ki67 in placenta from the vehicle and SCH groups at E18.5. Scale bars, 200 μm. **P < 0.01; Mann–Whitney U test. All data are presented as the mean ± SEM. Discussion The present study reported that YAP is abundantly expressed in the human placenta, but its phosphorylation is elevated in FGR-complicated pregnancies. Accordingly, known downstream transcriptional targets of YAP are downregulated in the FGR placenta. Moreover, in vitro experiments confirmed that p-YAP is upregulated by ERK inhibition in trophoblast cell lines. Downregulation of ERK impairs trophoblast invasion and migration but partly reverses these effects through YAP overexpression. In addition, the other FGR-like mouse model induced by ERK inhibition confirms the above findings. FGR is summarized as an end of various etiologies that mainly include maternal, placental, fetal, and genetic factors [50–52]. Among these factors, placental factors are the most supported [53, 54]. Appropriate fetal growth in utero largely depends on placental development and function, including nutrient transport and hormone supply. Dysfunction of trophoblasts in invasion, migration, and proliferation impairs spiral artery remodeling and placental perfusion, leading to undesirable placental function, which is especially associated with possible FGR pathogenesis. In our work, although fetal factors related to FGR development were not covered, influence from parents was taken into consideration during the process of date collection from parents. There was no significant difference between maternal age, weight, height, BMI, and their partner data in the FGR and normal groups, which indicated that the pregnancy outcome of FGR possibly originated from parents with low height or weight representing part of the genetic factors (Table S1). Nevertheless, the detailed mechanism underlying placental factors leading to FGR is still complex and diverse. Existing studies focused on placental factors of FGR vary. Taking recent research as examples, Lo et al. [55] suggested a novel mechanism for FGR relevance in dysregulation of factor glial cells missing 1 (GCM1) on trophoblast differentiation and function. Kohil et al. [56] reported that p45 nuclear factor erythroid derived 2 (NF-E2) negatively regulates the differentiation and apoptosis activation of human trophoblasts, which might be new biomarkers and/or therapies for FGR. The above studies revealed that the etiology of FGR is multifarious, and the reported mechanism might not represent pathophysiological relevance. The placenta is of great significance for fetal growth, and the placenta is an indispensable organ in utero. Our work attempted to discover some general characteristics of FGR through clinical samples and an FGR-like mouse model. YAP has a crucial role in organ/tissue development; therefore, this study investigated the correlation of YAP and FGR. YAP is a shuttling protein between the nucleus and plasma that also mediates such cell functions as invasion, migration, and angiogenesis. Phosphorylation of YAP results in cytoplasmic localization and functional inactivation. As expected, we showed that the FGR placenta is associated with downregulated expression of CTGF, CYR61, and AMOTL2, implying that phosphorylation-regulated subcellular localization of YAP is disturbed. CTGF, CYR61, and AMOTL2 are positive regulators of angiogenesis [57–59]. Previous studies demonstrated that placental angiogenesis leading to adequate placental perfusion is critical for placental and fetal development and provided an important condition for intrauterine fetal development. Cell migration is critical for blood vessel formation of the placenta and promotes the formation of blood vessel networks to ensure placental transport and metabolism. When placental trophoblast migration is impaired, as seen in FGR placenta, placental angiogenesis is also affected. Thus, impaired placental angiogenesis contributes to reduced placental vascular network formation, which results in fetal insufficiency and impaired gene expression of CTGF, CYR61, or AMOTL2 in trophoblast cells. To further determine the role of YAP in the development of the placenta, we profiled the expression pattern of placental YAP during gestation and found that phosphorylation of YAP increased along with the progression of gestation, suggesting that the growth of the placenta might be constrained by the translocation of YAP from the nucleus into the cytoplasm. Previous studies have suggested that YAP is critical for embryonic development in mouse implantation, but its association with placental development has not been determined. The results showed that YAP was mostly expressed in trophoblasts without altering the expression of total YAP. In addition, we also examined the subcellular localization of YAP in FGR placenta samples to pinpoint the phosphorylation of YAP in the placenta. Accordingly, the FGR placenta also exhibited more YAP export and less expression in the nucleus (Figure S3). This finding also implies that the phosphorylation of YAP and consequent removal of YAP from the nucleus might be a “brake” for excessive placental development, which is essential for favorable pregnancy outcomes. This finding could suggest that the pathogenesis of FGR might partially originate from abnormal p-YAP activation or aberrant high expression. Both MAPK/ERK and Hippo/YAP signaling have common effects on such processes as oncogenesis, cell proliferation, and differentiation. The regulatory role of ERK on YAP is not clear at present but has been suggested indirectly. Li et al. [59] reported that MEK, a direct upstream target of ERK, reduced phosphorylation by corresponding inhibitors, leading to impairment of both YAP expression and activity in hepatocarcinogenesis. You et al. showed that ERK inhibition by small interfering RNA (siRNA) or inhibitors downregulated the protein expression of YAP and, in turn, suppressed the transcriptional activity of the Hippo signal in non-small-cell lung cancer (NSCLC) [60]. Unlike the findings of these researchers, we did not observe the regulatory role of ERK on total YAP protein but its phosphorylation, which might be attributed to the inherent characteristics of trophoblasts and cancer cells. Rawat et al. reported that ERK inhibition-mediated elevated p-YAP was dependent on Mst1/2 homodimerization, which proceeded with the phosphorylation of YAP in HEK293 cells. Our further work on mRNA transcriptional analysis from HTR-8/SVneo and JAR trophoblast cell lines treated with ERKi (100 nM) showed that known YAP transcriptional downstream targets, CTGF, AMOTL2, and CYR61, were also regulated by ERK (Figure S4). To better address this discordance, further experiments relying on primary cells could be an important step toward the correlation of ERK and YAP. Previous studies demonstrated that the high incidence of FGR in developing countries is usually due to social reasons, such as gender violence, poor prepregnancy nutrition, low prepregnancy weights, and poverty [61, 62]. For this reason, a low nutrition diet-induced FGR-like animal model was developed to proceed with a series of studies of FGR pathogenesis [63]. Notably, this model only represented the causes of FGR by poverty or malnutrition. Another established FGR-like animal model induced by surgery, reduced uterine perfusion pressure (RUPP), could compensate for the existing deficiency in FGR animal studies, which served as a cause of insufficiency in placental supply [64]. To treat RUPP as an FGR model, one drawback was that researchers took this model as preeclampsia because of the elevated blood pressure in pregnant animals caused by clipping at the uterine artery. Our established FGR-like mouse model in this study was induced by intraperitoneal injection of an ERK inhibitor. As a result, placental p-ERK expression was significantly downregulated, while p-YAP was elevated. Notably, ERK-deficient mice showed abnormal placental development, and as a result, smaller placenta and fetuses were obtained. Nonetheless, intraperitoneal delivery of ERK inhibitors could result in a general knockdown of ERK expression in various animal organs or tissues, therefore, the observed phenotypes might not entirely originate from ERK deficiency in the placenta. To solve this limitation, placenta-targeted delivery can be achieved by bioengineering technology, such as engineered nanoparticles [65, 66]. Alternatively, placenta-specific ERK-deficient mice can be generated by Cre/loxP with a trophoblast-specific promoter [67]. These mentioned techniques could be notably helpful for extending our work on FGR. In conclusion, to the best of our knowledge, this study is the first to explore the pathological mechanism of FGR associated with YAP, which exhibits similar characteristics of tissue/organ restriction induced by YAP activity. We elucidated a putative mechanism of FGR pathogenesis in which aberrant activation of p-YAP results in trophoblast dysfunction. Furthermore, elucidating the mechanisms of YAP activity could provide a noninvasive intervention for the intrauterine treatment of FGR and warrants future study. Our results demonstrate a strong correlation of FGR with the activation status of YAP in placental and fetal development involving the regulation of ERK activity. Acknowledgments This study was funded by National Key R&D Program of China (2018YFC1004103), National Natural Science Foundation of China (81671488，81871189, 81520108013, and 81771613), and Chongqing Science and Technology Commission (cstc2017jcyjBX0045). We would like to acknowledge the support from the 111 Plan of the Ministry of Education of the People’ s Republic of China and the State Administration of Foreign Experts Affairs of China. Conflict of interest None. References 1. ACOG . ACOG practice bulletin no. 134: fetal growth restriction . Obstet Gynecol 2013 ; 121 : 1122 – 1133 . Crossref Search ADS PubMed WorldCat 2. Longo S , Bollani L, Decembrino L, Di Comite A, Angelini M, Stronati M. Short-term and long-term sequelae in intrauterine growth retardation (IUGR) . J Matern Fetal Neonatal Med 2013 ; 26 : 222 – 225 . Google Scholar Crossref Search ADS PubMed WorldCat 3. 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes Grant Support: This work was supported by the Ministry of Science and Technology of China (2018YFC1004103), the National Natural Science Foundation of China (81520108013, 81771613, 81671488, 81871189, 81901506), and Science and Technology Commission of Chongqing (cstc2017jcyjBX0045). Hao Wang, Ping Xu, Xiaofang Luo contributed equally to this work. © The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Phosphorylation of Yes-associated protein impairs trophoblast invasion and migration: implications for the pathogenesis of fetal growth restriction
JF - Biology of Reproduction
DO - 10.1093/biolre/ioaa112
DA - 2020-10-05
UR - https://www.deepdyve.com/lp/oxford-university-press/phosphorylation-of-yes-associated-protein-impairs-trophoblast-invasion-FVncLEDI8s
SP - 866
EP - 879
VL - 103
IS - 4
DP - DeepDyve
ER -