Downregulation of LncRNA-MEG3 promotes HTR8/SVneo cells apoptosis and attenuates its migration by repressing Notch1 signal in preeclampsia.
Wang Rongli,Zou Li
Reproduction (Cambridge, England)
A successful pregnancy crucially depends on well-regulated extravillous trophoblast migration and invasion. Maternally expressed gene 3 (MEG3) is a long noncoding RNA that plays an important role in regulating trophoblast cells cell function. As previously reported, the expression of MEG3 was reduced in preeclampsia, and downregulation of MEG3 could suppress trophoblast cells migration and promote its apoptosis. However, the downstream regulatory mechanism of MEG3 remains unknown. As reported, MEG3 could inhibit cell proliferation in endometrial carcinoma by regulating Notch signaling. Our previous studies have demonstrated that Notch1 is downregulated in preeclampsia and that inhibiting the expression of Notch1 could promote trophoblast cell apoptosis. Therefore, this study was designed to investigate the role of MEG3 and its the relationship with Notch1 in trophoblasts. In this study, the mRNA expression levels of both MEG3 and Notch1 were decreased in preeclampsia placenta (n = 15) compared to the normal samples (n = 15). Exogenous upregulation and downregulation of MEG3 in HTR8/SVneo cells were performed to investigate the role of MEG3 in cell biological behavior and its effects on Notch1 expression. The results showed that MEG3 enhancement promoted trophoblast cell migration and invasion and inhibited cell apoptosis. Downregulation of MEG3 elicited the opposite results. Associated factors, such as matrix metalloproteinases 2 (MMP2), BAX, and Bcl-2, were examined at the mRNA and protein levels. Our study demonstrated that MEG3 could regulate Notch1 expression to modulate trophoblast cell migration, invasion, and apoptosis, which may represent the molecular mechanism of poor placentation during preeclampsia.
CCN3 Signaling Is Differently Regulated in Placental Diseases Preeclampsia and Abnormally Invasive Placenta.
Duan Liyan,Schimmelmann Manuela,Wu Yuqing,Reisch Beatrix,Faas Marijke,Kimmig Rainer,Winterhager Elke,Köninger Angela,Gellhaus Alexandra
Frontiers in endocrinology
Objectives:An adequate development of the placenta includes trophoblast differentiation with the processes of trophoblast migration, invasion, cellular senescence and apoptosis which are all crucial to establishing a successful pregnancy. Altered placental development and function lead to placental diseases such as preeclampsia (PE) which is mainly characterized by insufficient trophoblast invasion and abnormally invasive placenta (AIP) disorders (, , or which are characterized by excessive trophoblast invasion. Both of them will cause maternal and fetal morbidity/mortality. However, the etiology of these diseases is still unclear. Our previous study has shown that the matricellular protein (NOV, CCN3) induces G0/G1 cell cycle arrest, drives trophoblast cells into senescence and activates FAK and Akt kinases resulting in reduced cell proliferation and enhanced migration capability of the human trophoblast cell line SGHPL-5. The present study focuses on whether CCN3 can alter cell cycle-regulated pathways associated with trophoblast senescence and invasion activity in pathological versus gestational age-matched control placentas. Methods:Cell cycle regulator proteins were investigated by immunoblotting and qPCR. For localization of CCN3, p16, p21, and Cyclin D1 proteins, co-immunohistochemistry was performed. Results:In early-onset PE placentas, CCN3 was expressed at a significantly lower level compared to gestational age-matched controls. The decrease of CCN3 level is associated with an increase in p53, Cyclin E1 and pRb protein expression, whereas the level of cleaved Notch-1, p21, Cyclin D1, pFAK, pAKT, and pmTOR protein decreased. In term AIP placentas, the expression of CCN3 was significantly increased compared to matched term controls. This increase was correlated to an increase in p53, p16, p21, Cyclin D1, cleaved Notch-1, pFAK, pAkt, and pmTOR whereas pRb was significantly decreased. However, in late PE and early AIP placentas, no significant differences in CCN3, p16, p21, Cyclin D1, p53, and cleaved Notch-1 expression were found when matched to appropriate controls. Conclusions:CCN3 expression levels are correlated to markers of cell cycle arrest oppositely in PE and AIP by activating the FAK/AKT pathway in AIP or down-regulating in PE. This may be one mechanism to explain the different pathological features of placental diseases, PE and AIP.
Forskolin promotes vasculogenic mimicry and invasion via Notch‑1‑activated epithelial‑to‑mesenchymal transition in syncytiolization of trophoblast cells in choriocarcinoma.
Xue Yan,Sun Rong,Zheng Wei,Yang Lei,An Ruifang
International journal of oncology
Choriocarcinoma (CC) is characterized by earlier blood metastasis compared with other female genital tumors and a high incidence of massive hemorrhage. Vasculogenic mimicry (VM) is highly associated with metastasis, and syncytiotrophoblast is involved in the formation of VM in CC. Forskolin is a typical activator of the cAMP pathway, which is involved in the syncytiolization of trophoblastic cells. In the present study, to determine the effects and mechanism of forskolin on cell invasion and VM during syncytiolization in vitro and in vivo, JEG‑3 and JAR cell lines were treated with 100 µM forskolin for 48 h, and wound healing and invasion assays were used to verify cell migratory and invasive capacities. A 3D culture and tube formation assays were established to detect VM. Variation of morphology and markers of the epithelial‑to‑mesenchymal transition (EMT) were assessed, and the role of the Notch signaling pathway was investigated in CC cells treated with forskolin. The results of the present study demonstrated that 100 µM forskolin induced syncytiolization of trophoblastic cells and enhanced the migratory and invasive abilities of JEG‑3 and JAR cell lines. In addition, the capacity of VM was significantly increased, whereas tube formation ability was decreased by forskolin in vitro and in vivo compared with the respective control groups. The cellular morphology exhibited EMT during the syncytiolization process, which was further supported by the changes in EMT marker expression, including downregulation of E‑cadherin and cytokeratin and upregulation of N‑cadherin, vimentin and zinc finger E‑box‑binding homeobox 1. The Notch‑1 signaling pathway was activated to induce EMT in forskolin‑induced VM process in CC cells, and VM and EMT could be reversed by using the γ‑secretase inhibitor DAPT to block the Notch‑1 pathway. Overall, the results of the present study demonstrated that forskolin enhanced the capacity of VM formation and metastasis through Notch‑1‑activated EMT in the syncytiolization of trophoblastic cells.
Curcumin stimulates angiogenesis through VEGF and expression of HLA-G in first-trimester human placental trophoblasts.
Basak Sanjay,Srinivas Vilasagaram,Mallepogu Aswani,Duttaroy Asim K
Cell biology international
Curcumin has a protective role in placental diseases like preeclampsia and preterm birth. Very little is known about its functional effects on growth, angiogenesis, and epigenetic activities of human first trimester placenta. HTR8/SVneo trophoblasts cells were used as model for human first trimester placenta. Effects of curcumin (≥80%) in these cells were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), radioactive thymidine uptake, quantitative real-time polymerase chain reaction (qRT-PCR), promoter DNA methylation, qRT-PCR array, tube formation, wound healing, and immunoblot assays. PC3 (prostate cancer), JEG-3 (trophoblast), and HMEC-1 (endothelial) cells were used as control in various experiments. Unlike in PC3 cells, curcumin stimulated growth, proliferation, and viability in HTR8/SVneo cells. Curcumin increased tube formation, and messenger RNA (mRNA) expression of angiogenic factors such as vascular endothelial growth factor A (VEGFA) and protein expression of proangiogenic factor VEGF receptor-2 and fatty acid-binding protein-4 (FABP4) in these cells. Curcumin-stimulated tube formation was associated with an increased expression of VEGFR2 and FABP4. The stimulatory effects of curcumin were inhibited by VEGFR2 (SU5416) and FABP4 (BMS309403) inhibitors. Curcumin also significantly increased both mRNA and protein expression of HLA-G in HTR8/SVneo cells. Curcumin increased mRNA expression of DNMT3A and NOTCH signaling system whereas down-regulated mRNA expression of HSD11β2. Curcumin enhanced hypomethylation of gene promoters against oxidative stress and DNA damage pathway mediators. Curcumin promotes cell growth, migration, and thus angiogenic potential of these cells. Increased expression of HLA-G by curcumin, hitherto unknown, is a novel finding since HLA-G not only favors the immune environment for invasive trophoblasts but also positively modulates angiogenesis.
miR-214-5p suppresses the proliferation, migration and invasion of trophoblast cells in pre-eclampsia by targeting jagged 1 to inhibit notch signaling pathway.
Gong Fengyan,Chai Wei,Wang Junwei,Cheng Huiyan,Shi Yuee,Cui Lifeng,Jia Guifeng
MicroRNA-214-5p has been reported to be expressed in placental tissue and suppressed the proliferation and invasion of various tumor cells. However, the role of miR-214-5p in pre-eclampsia has not been reported. We aimed to explore the effects of miR-214-5p in proliferation, migration, and invasion of placental trophoblast cells. RT-qPCR was used to quantify the miR-214-5p expression level in placental samples and four types of trophoblast cell lines. Cell proliferation was monitored by CCK-8 and Edu staining assays. Flow cytometry was used to determine the cell cycle. Wound healing and transwell assays were performed to measure the migratory and invasive capacities in JEG-3 and BEWO cells. In addition, we investigated whether miR-214-5p targeted Jagged 1 to regulate the Notch signaling pathway to affect trophoblast cells by luciferase assay and western blot. The expression of miR-214-5p was significantly increased in the placenta of patients with PE. Moreover, the proliferation, migration, and invasion of JEG-3 cells transfected with miR-214-5p mimic were inhibited. The results were reversed when BEWO cells were transfected with miR-214-5p inhibitor. The dual-luciferase assay demonstrated that miR-214-5p directly regulated Jagged 1. The expression of the proteins associated with the Notch signaling pathway, Jagged 1, Notch 1, HEY 1 and HES 1 were all decreased when Jagged 1 was negatively regulated by miR-214-5p. miR-214-5p directly down-regulated Jagged 1 expression, then suppressed proliferation, migration, and invasion of human placental trophoblast cells by inhibiting the Notch signaling pathway.
Circular RNA circ_0111277 attenuates human trophoblast cell invasion and migration by regulating miR-494/HTRA1/Notch-1 signal pathway in pre-eclampsia.
Ou Yuhua,Zhu Liqiong,Wei Xiangcai,Bai Shiyu,Chen Manqi,Chen Hui,Zhang Jianping
Cell death & disease
Mounting evidence has revealed that impaired spiral artery remodeling, placental dysfunction, and inadequate trophoblast invasion are closely correlated with the etiology and pathogenesis of pre-eclampsia (PE). Moreover, defective trophoblast invasion may trigger poor maternal-fetal circulation and placental hypoxia, leading to PE. However, the detailed molecular pathology of PE remains unclear. Although circRNAs, as a new type of stable and abundant endogenous noncoding RNA, have been proven to be essential to the pathogenesis of various diseases, their role in PE requires further verification. In this context, it is necessary to unveil the roles of circRNAs in regulating the migration and invasion of extravillous trophoblasts. In this study, using quantitative real-time PCR, we confirmed that hsa_circ_0111277 was upregulated in PE placentas relative to the level in normal pregnancy placentas. In addition, positive correlations between hsa_circ_0111277 expression and PE-related factors (proteinuria level at 24 h and placental weight) were identified by Pearson's analysis based on the clinical data of 25 PE patients. Moreover, fluorescence in situ hybridization analysis illustrated that circ_0111277 was preferentially localized within the cytoplasm. Mechanistically, circ_0111277 sponged hsa-miR-494-3p in trophoblast cells to attenuate the latter's repression by regulating HTRA1/Notch-1 expression. In conclusion, trophoblast cell migration and invasion were shown to be promoted and modulated by the hsa_circ_0111277/miR-494-3p/HTRA1/Notch-1 axis, which provides useful insight for exploring a new therapeutic approach for PE.
PreImplantation factor (PIF*) promotes embryotrophic and neuroprotective decidual genes: effect negated by epidermal growth factor.
Duzyj Christina M,Paidas Michael J,Jebailey Lellean,Huang Jing Shun,Barnea Eytan R
Journal of neurodevelopmental disorders
BACKGROUND:Intimate embryo-maternal interaction is paramount for pregnancy success post-implantation. The embryo follows a specific developmental timeline starting with neural system, dependent on endogenous and decidual factors. Beyond altered genetics/epigenetics, post-natal diseases may initiate at prenatal/neonatal, post-natal period, or through a continuum. Preimplantation factor (PIF) secreted by viable embryos promotes implantation and trophoblast invasion. Synthetic PIF reverses neuroinflammation in non-pregnant models. PIF targets embryo proteins that protect against oxidative stress and protein misfolding. We report of PIF's embryotrophic role and potential to prevent developmental disorders by regulating uterine milieu at implantation and first trimester. METHODS:PIF's effect on human implantation (human endometrial stromal cells (HESC)) and first-trimester decidua cultures (FTDC) was examined, by global gene expression (Affymetrix), disease-biomarkers ranking (GeneGo), neuro-specific genes (Ingenuity) and proteins (mass-spectrometry). PIF co-cultured epidermal growth factor (EGF) in both HESC and FTDC (Affymetrix) was evaluated. RESULTS:In HESC, PIF promotes neural differentiation and transmission genes (TLX2, EPHA10) while inhibiting retinoic acid receptor gene, which arrests growth. PIF promotes axon guidance and downregulates EGF-dependent neuroregulin signaling. In FTDC, PIF promotes bone morphogenetic protein pathway (SMAD1, 53-fold) and axonal guidance genes (EPH5) while inhibiting PPP2R2C, negative cell-growth regulator, involved in Alzheimer's and amyotrophic lateral sclerosis. In HESC, PIF affects angiotensin via beta-arrestin, transforming growth factor-beta (TGF-β), notch, BMP, and wingless-int (WNT) signaling pathways that promote neurogenesis involved in childhood neurodevelopmental diseases-autism and also affected epithelial-mesenchymal transition involved in neuromuscular disorders. In FTDC, PIF upregulates neural development and hormone signaling, while downregulating genes protecting against xenobiotic response leading to connective tissue disorders. In both HESC and FTDC, PIF affects neural development and transmission pathways. In HESC interactome, PIF promotes FUS gene, which controls genome integrity, while in FTDC, PIF upregulates STAT3 critical transcription signal. EGF abolished PIF's effect on HESC, decreasing metalloproteinase and prolactin receptor genes, thereby interfering with decidualization, while in FTDC, EGF co-cultured with PIF reduced ZHX2, gene that regulates neural AFP secretion. CONCLUSIONS:PIF promotes decidual trophic genes and proteins to regulate neural development. By regulating the uterine milieu, PIF may decrease embryo vulnerability to post-natal neurodevelopmental disorders. Examination of PIF-based intervention strategies used during embryogenesis to improve pregnancy prognosis and reduce post-natal vulnerability is clearly in order.
Decidualization induces a secretome switch in perivascular niche cells of the human endometrium.
Murakami Keisuke,Lee Yie Hou,Lucas Emma S,Chan Yi-Wah,Durairaj Ruban Peter,Takeda Satoru,Moore Jonathan D,Tan Bee K,Quenby Siobhan,Chan Jerry K Y,Gargett Caroline E,Brosens Jan J
The endometrial perivascular microenvironment is rich in mesenchymal stem-like cells that express type 1 integral membrane protein Sushi domain containing 2 (SUSD2) but the role of these cells in the decidual transformation of this tissue in pregnancy is unknown. We used an antibody directed against SUSD2 (W5C5) to isolate perivascular (W5C5(+)) and nonperivascular (W5C5(-)) fibroblasts from mid-luteal biopsies. We show that SUSD2 expression, and hence the ratio of W5C5(+):W5C5(-) cells, changes in culture depending on cell-cell contact and activation of the Notch signaling pathway. RNA sequencing revealed that cultures derived from W5C5(+) progenitor cells remain phenotypically distinct by the enrichment of novel and established endometrial perivascular signature genes. In an undifferentiated state, W5C5(+)-derived cells produced lower levels of various chemokines and inflammatory modulators when compared with their W5C5(-) counterparts. This divergence in secretomes was switched and became more pronounced upon decidualization, which transformed perivascular W5C5(+) cells into the dominant source of a range of chemokines and cytokines, including leukemia inhibitory factor and chemokine (C-C motif) ligand 7. Our findings suggest that the decidual response is spatially organized at the embryo-maternal interface with differentiating perivascular cells establishing distinct cytokine and chemokine profiles that could potentially direct trophoblast toward maternal vessels and govern local immune responses in pregnancy.
Notch and hippo converge on Cdx2 to specify the trophectoderm lineage in the mouse blastocyst.
Rayon Teresa,Menchero Sergio,Nieto Andres,Xenopoulos Panagiotis,Crespo Miguel,Cockburn Katie,Cañon Susana,Sasaki Hiroshi,Hadjantonakis Anna-Katerina,de la Pompa Jose Luis,Rossant Janet,Manzanares Miguel
The first lineage choice in mammalian embryogenesis is that between the trophectoderm, which gives rise to the trophoblast of the placenta, and the inner cell mass, from which is derived the embryo proper and the yolk sac. The establishment of these lineages is preceded by the inside-versus-outside positioning of cells in the early embryo and stochastic expression of key transcription factors, which is then resolved into lineage-restricted expression. The regulatory inputs that drive this restriction and how they relate to cell position are largely unknown. Here, we show an unsuspected role of Notch signaling in regulating trophectoderm-specific expression of Cdx2 in cooperation with TEAD4. Notch activity is restricted to outer cells and is able to influence positional allocation of blastomeres, mediating preferential localization to the trophectoderm. Our results show that multiple signaling inputs at preimplantation stages specify the first embryonic lineages.
Expression of notch family proteins in placentas from patients with early-onset severe preeclampsia.
Zhao Wei-Xiu,Huang Tao-Tao,Jiang Meng,Feng Ran,Lin Jian-Hua
Reproductive sciences (Thousand Oaks, Calif.)
OBJECTIVES:This study is aimed to identify the expression of Notch family proteins in placentas from patients with early-onset severe preeclampsia. STUDY DESIGN:The expression of Notch family proteins in placentas was investigated by immunohistochemistry, Western blotting, and real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS:The profile of distribution of all Notch family proteins in placentas from patients with early-onset severe preeclampsia is similar to that in normal placentas. All Notch family proteins are expressed in placental trophoblasts. Moreover, Notch1 and Jagged1 (Jag1) are detected in placental endothelial cells. Real-time RT-PCR showed that messenger RNA levels of Notch2 and Delta-like4 (Dll4) in placentas from patients with early-onset severe preeclampsia are lower than that of normal placentas. Western blotting showed a significant increase in Notch3 expression and a significant decrease in Notch2 expression in placentas from patients with early-onset severe preeclampsia relative to those in normal placentas. CONCLUSION:The results suggest that Notch2 and Notch3 may play some roles in the pathogenesis of preeclampsia.
Dysregulation of LncRNAs in Placenta and Pathogenesis of Preeclampsia.
Song Xuejing,Luo Xiucui,Gao Quansheng,Wang Yuhuan,Gao Qing,Long Wei
Current drug targets
BACKGROUND:Preeclampsia, a gestational disease characterized by hypertension and proteinuria twenty weeks into pregnancy, is one of the leading causes of fetal and maternal mortality. Although multiple genetic and environmental factors are found to be related to the preeclampsia risk, the pathogenic pathways remain largely undefined. The placenta plays a critical role in the fetal development by carrying out the barrier, fetal-maternal exchange, and endocrine functions during pregnancy. Accumulated data indicated that the expression of multiple long noncoding RNA (LncRNA) is dysregulated in preeclamptic placentas. Moreover, manipulation of LncRNA expression led to functional alterations in trophoblast cell cultures, including changes in proliferation, differentiation, apoptosis, and migration. OBJECTIVE:This article reviews published data on this subject and provides detailed information on the regulation and function of LncRNAs IGF2/H19, MEG3, SPRY4-IT1, HOTAIR, MALAT1, and FLT1P1 and CEACAMP8 in placental trophoblasts. The potential mechanisms underlying the action of these LncRNAs are also discussed to facilitate a better understanding on the potential role of these LncRNAs for the pathogenesis of preeclampsia. CONCLUSION:It is elaborated that some lncRNAs probably contribute to the pathogenesis of preeclampsia through methylation, Notch-EGFL7 signaling pathway and Wnt/β-catenin pathway.
Notch2 and Notch3 suppress the proliferation and mediate invasion of trophoblast cell lines.
Zhao Wei-Xiu,Wu Zhen-Ming,Liu Wei,Lin Jian-Hua
Notch signaling pathways play important roles in cell fate and many diseases, including preeclampsia, the dysregulation of which may be the main cause of maternal mortality. This study aimed to investigate the roles of Notch2 and Notch3 in proliferation and invasion in trophoblast cell lines (BeWo and JAR). Small hairpin RNAs targeting Notch2/Notch3 and Notch2/Notch3-overexpression vectors were designed, constructed and transfected into BeWo and JAR cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were then used to detect Notch2 and Notch3 mRNA and protein levels, and confirm the efficiency of silence and overexpression. Flow cytometry assays were conducted to evaluate the cell cycle of the two cell lines, and transwell assays were used to detect migration and invasion. Western blot analysis was also performed to show the alteration of the cell lines' physiological activities at protein level.When Notch2 was downregulated in BeWo cells, proliferation was dramatically promoted, while migration and invasion were significantly inhibited. When Notch2 was upregulated in JAR cells, proliferation was inhibited, but migration and invasion were promoted. After overexpression of Notch3 in BeWo cells, proliferation was downregulated, but migration and invasion were both upregulated. By contrast, the silencing of Notch3 expression in JAR cells significantly enhanced proliferation, but suppressed migration and invasion. These data indicated that Notch2 and Notch3 mediate the invasion and migration of BeWo and JAR cells, and may play a potential role in early onset severe preeclampsia.
RITA Is Expressed in Trophoblastic Cells and Is Involved in Differentiation Processes of the Placenta.
Wildner Julia Maria,Friemel Alexandra,Jennewein Lukas,Roth Susanne,Ritter Andreas,Schüttler Cornelia,Chen Qi,Louwen Frank,Yuan Juping,Kreis Nina-Naomi
Preeclampsia (PE) remains a leading cause of maternal and perinatal mortality and morbidity worldwide. Its pathogenesis has not been fully elucidated and no causal therapy is currently available. It is of clinical relevance to decipher novel molecular biomarkers. RITA (BP-J (recombination signal binding protein J)-nteracting and ubulin-ssociated protein) has been identified as a negative modulator of the Notch pathway and as a microtubule-associated protein important for cell migration and invasion. In the present work, we have systematically studied RITA's expression in primary placental tissues from patients with early- and late-onset PE as well as in various trophoblastic cell lines. RITA is expressed in primary placental tissues throughout gestation, especially in proliferative villous cytotrophoblasts, in the terminally differentiated syncytiotrophoblast, and in migrating extravillous trophoblasts. 's messenger RNA (mRNA) level is decreased in primary tissue samples from early-onset PE patients. The deficiency of RITA impairs the motility and invasion capacity of trophoblastic cell lines, and compromises the fusion ability of trophoblast-derived choriocarcinoma cells. These data suggest that RITA may play important roles in the development of the placenta and possibly in the pathogenesis of PE.
Alteration of Delta-like ligand 1 and Notch 1 receptor in various placental disorders with special reference to early onset preeclampsia.
Shimanuki Yota,Mitomi Hiroyuki,Fukumura Yuki,Makino Shintaro,Itakura Atsuo,Yao Takashi,Takeda Satoru
Notch signaling pathway has been shown to be dysregulated in placentas with preeclampsia, but there has been a lack of studies on methylation of Notch family genes in this disorder. We therefore executed methylation-specific polymerase chain reaction and immunostaining for Notch 1 receptor and the activating ligand, Delta-like (DLL) 1, with placental tissues from cases of preeclampsia (early onset, n = 18; late-onset, n = 19) and other placental disorders, including maternal complications such as diabetes mellitus and collagen disease (n = 10), fetal growth restriction (n = 17), fetal anomaly (n = 23), preterm birth (n = 15), miscarriage (n = 25), and hydatidiform moles (n = 9) as well as term births (n = 12). The frequency of DLL1 methylation was higher in early onset preeclamptic placentas (61%) than the other subjects (0%-36%; P ≤ .016). Appreciable samples (36%) of miscarriage also represented DLL1 methylation. None of the samples studied showed Notch 1 methylation. On gestational period-matched analysis, the rate of DLL1 methylation was higher in early onset preeclampsia (83.3%) than preterm birth (13.3%; P < .001), with no significant differences in clinical backgrounds between the two. In this analysis, increase of syncytial knots and accelerated villous maturation were most prominent in DLL1-methylated placentas with early onset preeclampsia. Notch 1 and DLL1 expressions in villous trophoblasts and endothelial cells were significantly lower in early onset preeclamptic placentas as compared with preterm birth controls. In conclusion, altered Notch signaling via methylation of DLL1 is likely involved in possible disease-related mechanisms of early onset preeclampsia. Alternatively, DLL1 methylation in early onset preeclampsia could be a manifestation of a lack of placental maturation, similar to miscarriage.
Notch Signaling in Inflammation-Induced Preterm Labor.
Jaiswal Mukesh K,Agrawal Varkha,Pamarthy Sahithi,Katara Gajendra K,Kulshrestha Arpita,Gilman-Sachs Alice,Beaman Kenneth D,Hirsch Emmet
Notch signaling plays an important role in regulation of innate immune responses and trophoblast function during pregnancy. To identify the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligands (DLL (Delta-like protein)-1/3/4), Jagged 1/2) and Notch-induced transcription factor Hes1 were assessed during preterm labor. Preterm labor was initiated on gestation day 14.5 by intrauterine (IU) injection of peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C). Notch1, Notch2, Notch4, DLL-1 and nuclear localization of Hes1 were significantly elevated in uterus and placenta during PGN+poly(I:C)-induced preterm labor. Ex vivo, Gamma secretase inhibitor (GSI) (inhibitor of Notch receptor processing) significantly diminished the PGN+poly(I:C)-induced secretion of M1- and M2-associated cytokines in decidual macrophages, and of proinflammatory cytokines (IFN-γ, TNF-α and IL-6) and chemokines (MIP-1β) in decidual and placental cells. Conversely, angiogenesis factors including Notch ligands Jagged 1/2 and DLL-4 and VEGF were significantly reduced in uterus and placenta during PGN+poly(I:C)-induced preterm labor. In vivo GSI treatment prevents PGN+poly(I:C)-induced preterm delivery by 55.5% and increased the number of live fetuses in-utero significantly compared to respective controls 48 hrs after injections. In summary, Notch signaling is activated during PGN+poly(I:C)-induced preterm labor, resulting in upregulation of pro-inflammatory responses, and its inhibition improves in-utero survival of live fetuses.
LncRNA TDRG1/miR-214-5p axis affects preeclampsia by modulating trophoblast cells.
Gong Fengyan,Cheng Huiyan,Shi Yuee,Cui Lifeng,Jia Guifeng
Cell biochemistry and function
Because of limited treatment options, preeclampsia (PE) is the leading cause of perinatal morbidity and mortality worldwide. Recently, lncRNA TDRG1 is reported to be aberrantly down-regulated in PE placenta, and the abnormal expression of TDRG1 might play a key or partial role in PE development. In this study, we found that TDRG1 was significantly down-regulated in PE placenta compared with the normal placenta. The cell proliferation, migration, invasion, and cell cycle were explored by CCK-8, wound-healing, transwell, and flow cytometer assay, respectively. Experimental results showed that TDRG1 accelerated the proliferation, migration, and invasion of trophoblast cells. Dual-luciferase reporter assays confirmed that TDRG1 could bind to miR-214-5p. Besides, knockdown of TDRG1 suppressed the cell proliferation, migration, and invasion, while knockdown of miR-214-5p reversed the effect. Jagged1 and Notch1 were negatively regulated by miR-214-5p while positively modulated by TDRG1. In conclusion, TDRG1 promoted trophoblast cells viability and invasion by negatively regulating miR-214-5p expression, contributing to a better understanding of PE pathogenesis and providing new light on TDRG1-directed diagnosis and treatment. SIGNIFICANCE OF THE STUDY: In this work, we observed that TDRG1 was able to promote cell proliferation, migration, and invasion cells by suppressing the expression of miR-214-5p and regulating the Notch signalling pathway in trophoblast cells. As far as we know, the effect of TDRG1/miR-214-5p axis on cell viability, migration, and invasion of trophoblast cells was firstly introduced. Our findings provided a better understanding of the mechanism of PE. Moreover, it is reasonable to believe that TDRG1 may be employed as a strategy to treat PE in the future.
Notch1 signaling antagonizes transforming growth factor-β pathway and induces apoptosis in rabbit trophoblast stem cells.
Tan Tao,Lu Bin,Zhang Jing,Niu Yuyu,Si Wei,Wei Qiang,Ji Weizhi
Stem cells and development
During mammalian development, placental growth needs to be tightly controlled by apoptosis. However, despite the potentially significant problems, the strategies used to balance growth and apoptosis have remained elusive. Here we report that activation of the Notch1 signal pathway inhibits transduction of transforming growth factor (TGF)-β signaling, which leads to cell cycle arrest and apoptosis in rabbit trophoblast stem cells (TSCs). The subcellular location of notch intracellular domain 1 (NICD1) appears to determine whether TGF-β signaling will be inhibited or not. Moreover, changes in NICD1 subcellular location are regulated by intracellular calcium distribution. Collectively, these results establish a potential mechanism whereby TSCs can balance growth and apoptosis, and thus guarantee the development of the fetus.
Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast.
Plessl K,Haider S,Fiala C,Pollheimer J,Knöfler M
INTRODUCTION:Notch signalling has been shown to control cytotrophoblast (CTB) proliferation, differentiation and motility suggesting that the conserved signalling pathway could be critical for human placental development. Since individual Notch receptors have not been elucidated, we herein investigated expression pattern and function of Notch2 in different first trimester trophoblast subpopulations. METHODS:Localisation of Notch2 was analysed in first trimester placental and decidual tissues using immunofluorescence. Notch2 transcript and protein levels were studied by qRT-PCR and Western blotting in proliferative EGF receptor (EGFR)(+) and differentiated HLA-G(+) CTBs, respectively, isolated from early placentae by MACS. CTB migration through fibronectin-coated transwells as well as proliferation (EdU labelling) in floating villous explant cultures and primary CTBs were investigated in the presence of Notch2 siRNAs or specific antibodies blocking Notch2 cleavage. RESULTS:In tissue sections Notch2 expression was higher in HLA-G(+) distal cell column trophoblasts (dCCTs) compared to proximal CCTs. Accordingly, expression of Notch2 mRNA and protein were elevated in isolated HLA-G(+) CTBs compared to EGFR(+) CTBs. Notch2 was also detectable in interstitial CTBs as well as in intramural CTBs associated with maternal decidual vessels. Antibody-mediated inhibition of Notch2 signalling did not affect proliferation, but increased migration of SGHPL-5 cells and primary CTBs. Similarly, Notch2 siRNA treatment promoted trophoblast motility. DISCUSSION:Notch2 is present in differentiated cells of the extravillous trophoblast lineage, such as dCCTs, interstitial and intramural CTBs, suggesting diverse roles of the particular receptor. Notch2 signalling, activated by cell-cell contact of neighbouring dCCTs, could attenuate trophoblast migration.
Notch and Hippo signaling converge on Strawberry Notch 1 (Sbno1) to synergistically activate Cdx2 during specification of the trophectoderm.
Watanabe Yusuke,Miyasaka Kota Y,Kubo Atsushi,Kida Yasuyuki S,Nakagawa Osamu,Hirate Yoshikazu,Sasaki Hiroshi,Ogura Toshihiko
The first binary cell fate decision occurs at the morula stage and gives rise to two distinct types of cells that constitute the trophectoderm (TE) and inner cell mass (ICM). The cell fate determinant, Cdx2, is induced in TE cells and plays an essential role in their differentiation and maintenance. Notch and Hippo signaling cascades are assumed to converge onto regulatory elements of Cdx2, however, the underlying molecular mechanisms are largely unknown. Here, we show involvement of Strawberry Notch1 (Sbno1), a novel chromatin factor of the helicase superfamily 2, during preimplantation development. Sbno1 knockout embryos die at the preimplantation stage without forming a blastocoel, and Cdx2 is not turned on even though both Yap and Tead4 reside normally in nuclei. Accordingly, Sbno1 acts on the trophectoderm-enhancer (TEE) of Cdx2, ensuring its robust and synergistic activation by the Yap/Tead4 and NICD/Rbpj complexes. Interestingly, this synergism is enhanced when cells are mechanically stretched, which might reflect that TE cells are continuously stretched by the expanding ICM and blastocoel cavity. In addition, the histone chaperone, FACT (FAcilitates Chromatin Transcription) physically interacts with Sbno1. Our data provide new evidence on TE specification, highlighting unexpected but essential functions of the highly conserved chromatin factor, Sbno1.
Distinct mechanisms regulate Cdx2 expression in the blastocyst and in trophoblast stem cells.
Rayon Teresa,Menchero Sergio,Rollán Isabel,Ors Inmaculada,Helness Anne,Crespo Miguel,Nieto Andres,Azuara Véronique,Rossant Janet,Manzanares Miguel
The first intercellular differences during mammalian embryogenesis arise in the blastocyst, producing the inner cell mass and the trophectoderm. The trophectoderm is the first extraembryonic tissue and does not contribute to the embryo proper, its differentiation instead forming tissues that sustain embryonic development. Crucial roles in extraembryonic differentiation have been identified for certain transcription factors, but a comprehensive picture of the regulation of this early specification is still lacking. Here, we investigated whether the regulatory mechanisms involved in Cdx2 expression in the blastocyst are also utilized in the postimplantation embryo. We analyzed an enhancer that is regulated through Hippo and Notch in the blastocyst trophectoderm, unexpectedly finding that it is inactive in the extraembryonic structures at postimplantation stages. Further analysis identified other Cdx2 regulatory elements including a stem-cell specific regulatory sequence and an element that drives reporter expression in the trophectoderm, a subset of cells in the extraembryonic region of the postimplantation embryo and in trophoblast stem cells. The cross-comparison in this study of cis-regulatory elements employed in the blastocyst, stem cell populations and the postimplantation embryo provides new insights into early mammalian development and suggests a two-step mechanism in Cdx2 regulation.
Effects of Notch2 and Notch3 on Cell Proliferation and Apoptosis of Trophoblast Cell Lines.
Zhao Wei-Xiu,Zhuang Xu,Huang Tao-Tao,Feng Ran,Lin Jian-Hua
International journal of medical sciences
AIMS:To investigate the effect of Notch2 and Notch3 on cell proliferation and apoptosis of two trophoblast cell lines, BeWo and JAR. METHODS:Notch2 and Notch3 expression in BeWo and JAR cells was upregulated or downregulated using lentivirus-mediated overexpression or RNA interference. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. The effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V-PE Apoptosis kit. Lentivirus-based overexpression vectors were constructed by cloning the full-length coding sequences of human Notch2 and Notch3 C-terminally tagged with GFP or GFP alone (control) into a lentivirus-based expression vector. Lentivirus-based gene silencing vectors were prepared by cloning small interfering sequences targeting human Notch2 and Notch3 and scrambled control RNA sequence into a lentivirus-based gene knockdown vector. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. And the effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V PE Apoptosis kit. RESULTS:We found that the downregulation of Notch2 and Notch3 gene expression in BeWo and JAR cells resulted in an increase in cell proliferation, while upregulation of Notch3 and Notch2 expression led to a decrease in cell proliferation. Moreover, the overexpression of Notch3 and Notch2 in BeWo and JAR cells reduced apoptosis in these trophoblast cell lines, whereas apoptosis was increased in the cells in which the expression of Notch3 and Notch2 was downregulated. CONCLUSIONS:Notch2 and Notch3 inhibited both cell proliferation and cell apoptosis in BeWo and JAR trophoblast cell lines.
Notch signaling plays a critical role in motility and differentiation of human first-trimester cytotrophoblasts.
Haider Sandra,Meinhardt Gudrun,Velicky Philipp,Otti Gerlinde R,Whitley Guy,Fiala Christian,Pollheimer Jürgen,Knöfler Martin
Failures in human extravillous trophoblast (EVT) development could be involved in the pathogenesis of pregnancy diseases. However, the underlying mechanisms have been poorly characterized. Here, we provide evidence that Notch signaling could represent a key regulatory pathway controlling trophoblast proliferation, motility, and differentiation. Immunofluorescence of first-trimester placental tissues revealed expression of Notch receptors (Notch2 and Notch3) and membrane-anchored ligands (delta-like ligand [DLL] 1 and -4 and Jagged [JAG] 1 and -2) in villous cytotrophoblasts (vCTBs), cell column trophoblasts (CCTs), and EVTs. Notch4 and Notch1 were exclusively expressed in vCTBs and in CCTs, respectively. Both proteins decreased in Western blot analyses of first-trimester, primary cytotrophoblasts (CTBs) differentiating on fibronectin. Luciferase reporter analyses suggested basal, canonical Notch activity in SGHPL-5 cells and primary cells that was increased upon seeding on DLL4-coated dishes and diminished in the presence of the Notch/γ-secretase inhibitors N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) or L-685,458. Bromodeoxyuridine labeling, cyclin D1 mRNA expression, and cell counting indicated that chemical inhibition of Notch signaling elevated proliferation in the different primary trophoblast model systems. Notch inhibition also increased motility of SGHPL-5 cells through uncoated and fibronectin-coated Transwells, motility of primary CTBs, as well as migration in villous explant cultures on collagen I. Accordingly, small interfering RNA-mediated gene silencing of Notch1 also elevated SGHPL-5 cell migration. In contrast, motility of primary cultures and SGHPL-5 cells was diminished in the presence of DLL4. Moreover, DAPT increased markers of differentiated EVT, ie, human leukocyte antigen G1, integrin α5, and T-cell factor 4, whereas DLL4 provoked the opposite. In summary, the data suggest that canonical Notch signaling impairs motility and differentiation of first-trimester CTBs.
G9a controls placental vascular maturation by activating the Notch Pathway.
Chi Lijun,Ahmed Abdalla,Roy Anna R,Vuong Sandra,Cahill Lindsay S,Caporiccio Laura,Sled John G,Caniggia Isabella,Wilson Michael D,Delgado-Olguin Paul
Development (Cambridge, England)
Defective fetoplacental vascular maturation causes intrauterine growth restriction (IUGR). A transcriptional switch initiates placental maturation, during which blood vessels elongate. However, the cellular mechanisms and regulatory pathways involved are unknown. We show that the histone methyltransferase G9a, also known as Ehmt2, activates the Notch pathway to promote placental vascular maturation. Placental vasculature from embryos with G9a-deficient endothelial progenitor cells failed to expand owing to decreased endothelial cell proliferation and increased trophoblast proliferation. Moreover, G9a deficiency altered the transcriptional switch initiating placental maturation and caused downregulation of Notch pathway effectors including Importantly, Notch pathway activation in G9a-deficient endothelial progenitors extended embryonic life and rescued placental vascular expansion. Thus, G9a activates the Notch pathway to balance endothelial cell and trophoblast proliferation and coordinates the transcriptional switch controlling placental vascular maturation. Accordingly, and were downregulated in human placentae from IUGR-affected pregnancies, suggesting that G9a is an important regulator in placental diseases caused by defective vascular maturation.
Novel expression of EGFL7 in placental trophoblast and endothelial cells and its implication in preeclampsia.
Lacko Lauretta A,Massimiani Micol,Sones Jenny L,Hurtado Romulo,Salvi Silvia,Ferrazzani Sergio,Davisson Robin L,Campagnolo Luisa,Stuhlmann Heidi
Mechanisms of development
The mammalian placenta is the site of nutrient and gas exchange between the mother and fetus, and is comprised of two principal cell types, trophoblasts and endothelial cells. Proper placental development requires invasion and differentiation of trophoblast cells, together with coordinated fetal vasculogenesis and maternal vascular remodeling. Disruption in these processes can result in placental pathologies such as preeclampsia (PE), a disease characterized by late gestational hypertension and proteinuria. Epidermal Growth Factor Like Domain 7 (EGFL7) is a largely endothelial-restricted secreted factor that is critical for embryonic vascular development, and functions by modulating the Notch signaling pathway. However, the role of EGFL7 in placental development remains unknown. In this study, we use mouse models and human placentas to begin to understand the role of EGFL7 during normal and pathological placentation. We show that Egfl7 is expressed by the endothelium of both the maternal and fetal vasculature throughout placental development. Importantly, we uncovered a previously unknown site of EGFL7 expression in the trophoblast cell lineage, including the trophectoderm, trophoblast stem cells, and placental trophoblasts. Our results demonstrate significantly reduced Egfl7 expression in human PE placentas, concurrent with a downregulation of Notch target genes. Moreover, using the BPH/5 mouse model of PE, we show that the downregulation of Egfl7 in compromised placentas occurs prior to the onset of characteristic maternal signs of PE. Together, our results implicate Egfl7 as a possible factor in normal placental development and in the etiology of PE.
Polychlorinated biphenyls target Notch/Dll and VEGF R2 in the mouse placenta and human trophoblast cell lines for their anti-angiogenic effects.
Kalkunte Satyan,Huang Zheping,Lippe Eliana,Kumar Sunil,Robertson Larry W,Sharma Surendra
The intrauterine environment is particularly vulnerable to environmental exposures. We previously established a mouse model that provided evidence for pregnancy complications and placental anti-angiogenesis in response to Aroclor 1254 (A-1254), a mixture of polychlorinated biphenyls (PCBs). Importantly, these effects were observed in IL-10, but not wild type, mice, suggesting that IL-10 deficiency predisposes to pregnancy disruptive effects of environmental toxicants. However, the mechanisms by which PCBs cause anti-angiogenic effects are unclear. Here, we evaluated PCB-mediated anti-angiogenic effects by diverse but complementary approaches, including HUVEC-mediated trophoblast invasion in nude mice, in vitro three-dimensional capillary tube formation involving HUVEC and/or HTR8 trophoblasts, and aortic ring endothelial cell outgrowth/sprouting. Taken together, our data suggest that PCBs act as potent anti-angiogenic agents. Importantly, we show that treatment of pregnant IL-10 mice with A-1254 resulted in placental activation of the Notch/Delta-like ligand (Dll) pathway, a master regulator of cell-cell interaction and vascular patterning. Similar results were obtained with HUVEC and HTR8 trophoblasts. Rescue of A-1254-induced disruption of HUVEC-based tube formation by γ-secretase inhibitor L1790 confirmed the critical role of the Notch/Dll pathway. Our data suggest that PCBs impart pregnancy disruptive functions by activating the Notch/Dll pathway and by inducing anti-angiogenic effects at the maternal-fetal interface.
Total flavonoids from Semen Cuscutae target MMP9 and promote invasion of EVT cells via Notch/AKT/MAPK signaling pathways.
Gao Feixia,Zhou Chun,Qiu Weiyu,Wu Haiwang,Li Jing,Peng Jinting,Qiu Min,Liang Chun,Gao Jie,Luo Songping
Miscarriage is a common condition during pregnancy and its mechanisms remain largely unknown. Extravillous trophoblast (EVT) cell invasion is required to maintain normal pregnancy and its malfunction has been proposed as a major cause for miscarriage. Homeostasis of matrix metalloproteinase 9 (MMP9) is a key to regulate EVT cell invasion. Total flavonoids from Semen Cuscutae (TFSC) have been applied clinically used for preventing or treating miscarriage in the past. Given its potential clinical benefit on preventing miscarriage, this study aims at examining the therapeutic effect of TFSC in the prevention of premature birth by upregulating MMP9 and promote EVT cell invasion. HTR-8 cells migration and invasion functions were analyzed using wound healing and transwell assays. The regulatory effect of TFSC on MMP9 expression and relevant signaling pathways were analyzed by Western Blot. The results show compared to control group, TFSC significantly promoted the migration of EVT cells in a dose and time-dependent manner. The migration and invasion of EVT cells were maximized at the highest dosage of 5 μg/ml of TFSC. The expression of MMP9 in EVT cells was significantly increased after TFSC treatment. Furthermore, cells treated with TFSC significantly upregulated protein expressions in Notch, AKT and p38/MAPK signaling pathways. We believe TFSC can promote the migration and invasion of EVT cells by increasing MMP9 expression, and prevent miscarriage by activating Notch, AKT, and MAPK signaling pathways.
Cellular growth and tube formation of HTR8/SVneo trophoblast: effects of exogenously added fatty acid-binding protein-4 and its inhibitor.
Basak Sanjay,Sarkar Arnab,Mathapati Santosh,Duttaroy Asim K
Molecular and cellular biochemistry
Adequate placental angiogenesis is critical for the establishment of the placental circulation and thus for normal feto-placental growth and development. Fatty acid-binding protein-4 (FABP4) plays a pro-angiogenic role in endothelial cells; however, very little information is available in placental first trimester trophoblast cells. Here we report that exogenously added FABP4 (exo-FABP4) stimulated tube formation (as a measure of in vitro angiogenesis) in HTR8/SVneo trophoblastic cells. HTR-8/SVneo cells were incubated in the presence of exogenously added FABP4 at different concentrations and time points. Cellular growth, proliferation, in vitro tube formation, expression of growth stimulatory-, fatty acid transporters, and angiogenic genes were investigated. Internalization of exo-FABP4 was carried out using immunocytochemistry. Radioactive fatty acid uptake was determined in the presence and absence of FABP4 metabolic inhibitor. Exo-FABP4 (10-100 ng/ml) stimulated proliferation of HTR8/SVneo cells as compared to control. Exo-FABP4 dose dependently increased growth and viability of the cells to the similar extent as done by 50 µM of arachidonic acid. Exo-FABP4-induced tube formation and proliferation were significantly inhibited by FABP4 (BMS309403) inhibitor. Exo-FABP4 stimulated the expression of growth stimulatory genes such as tissue inhibitor of matrix metalloproteinases-1 (TIMP1), insulin-like growth factor 1 (IGF1), and also prokineticin 2 (PROK2), the pro-angiogenic mediators in these cells. In addition, expressions of genes associated with proliferation and differentiation such as sonic hedgehog (SHH) and WNT1 inducible signalling pathway protein 1 (WISP1) were significantly expressed when cells were exposed to exo-FABP4. Our findings reveal a pro-angiogenic role of FABP4 in first trimester placental trophoblast cells and its regulation may have impact in placental physiology.
Integrin α2 marks a niche of trophoblast progenitor cells in first trimester human placenta.
Lee Cheryl Q E,Turco Margherita Y,Gardner Lucy,Simons Benjamin D,Hemberger Myriam,Moffett Ashley
Development (Cambridge, England)
During pregnancy the trophoblast cells of the placenta are the only fetal cells in direct contact with maternal blood and decidua. Their functions include transport of nutrients and oxygen, secretion of pregnancy hormones, remodelling of the uterine arteries, and communicating with maternal cells. Despite the importance of trophoblast cells in placental development and successful pregnancy, little is known about the identity, location and differentiation of human trophoblast progenitors. We identify a proliferative trophoblast niche at the base of the cytotrophoblast cell columns in first trimester placentas that is characterised by integrin α2 (ITGA2) expression. Pulse-chase experiments with 5-iodo-2'-deoxyuridine indicate that these cells might contribute to both villous (VCT) and extravillous (EVT) lineages. These proliferating trophoblast cells can be isolated by flow cytometry using ITGA2 as a marker and express genes from both VCT and EVT. Microarray expression analysis shows that ITAG2 cells display a unique transcriptional signature, including genes involved in NOTCH signalling, and exhibit a combination of epithelial and mesenchymal characteristics. ITGA2 thus marks a niche allowing the study of pure populations of trophoblast progenitor cells.
Down-regulation of transcription factor OVOL2 contributes to epithelial-mesenchymal transition in a noninvasive type of trophoblast implantation to the maternal endometrium.
Bai Rulan,Kusama Kazuya,Nakamura Keigo,Sakurai Toshihiro,Kimura Koji,Ideta Atsushi,Aoyagi Yoshito,Imakawa Kazuhiko
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Embryo implantation into the uterine endometrium is required for pregnancy establishment in most mammals. By using global expression analysis, we investigated the molecules that are related to epithelial-mesenchymal transition (EMT) in noninvasive bovine trophoblasts and found that the transcription factor, ovo-like zinc finger 2 ( OVOL2), which is essential for mesenchymal-epithelial transition in various cancers, was down-regulated after trophoblast attachment to the endometrial epithelium in utero. In cultured bovine trophoblast cells, OVOL2 down-regulation occurred only when cells were allowed to attach to bovine endometrial epithelial cells via the TEAD3/YAP signaling pathway. This resulted in the up-regulation of the EMT-associated transcription factors, ZEB1 and SNAI2, and the mesenchymal cell markers, N-cadherin ( CDH2) and vimentin ( VIM), whereas epithelial cell marker, E-cadherin ( CDH1), was down-regulated. In contrast, OVOL2 overexpression in bovine trophoblast cells exhibited a decrease in ZEB1 transcripts and an increase in E-cadherin. These observations revealed that ovo-like protein (OVOL)2 down-regulation occurred concurrently with conceptus implantation into the uterine endometrium via the YAP/TEAD3 signaling pathway, and suggest that the down-regulation of OVOL2 expression contributes to the up-regulation of EMT-related transcription factor expression, which enables EMT progression in the noninvasive bovine trophectoderm postimplantation.-Bai, R., Kusama, K., Nakamura, K., Sakurai, T., Kimura, K., Ideta, A., Aoyagi, Y., Imakawa, K. Down-regulation of transcription factor OVOL2 contributes to epithelial-mesenchymal transition in a noninvasive type of trophoblast implantation to the maternal endometrium.
Human placenta and trophoblast development: key molecular mechanisms and model systems.
Knöfler Martin,Haider Sandra,Saleh Leila,Pollheimer Jürgen,Gamage Teena K J B,James Joanna
Cellular and molecular life sciences : CMLS
Abnormal placentation is considered as an underlying cause of various pregnancy complications such as miscarriage, preeclampsia and intrauterine growth restriction, the latter increasing the risk for the development of severe disorders in later life such as cardiovascular disease and type 2 diabetes. Despite their importance, the molecular mechanisms governing human placental formation and trophoblast cell lineage specification and differentiation have been poorly unravelled, mostly due to the lack of appropriate cellular model systems. However, over the past few years major progress has been made by establishing self-renewing human trophoblast stem cells and 3-dimensional organoids from human blastocysts and early placental tissues opening the path for detailed molecular investigations. Herein, we summarize the present knowledge about human placental development, its stem cells, progenitors and differentiated cell types in the trophoblast epithelium and the villous core. Anatomy of the early placenta, current model systems, and critical key regulatory factors and signalling cascades governing placentation will be elucidated. In this context, we will discuss the role of the developmental pathways Wingless and Notch, controlling trophoblast stemness/differentiation and formation of invasive trophoblast progenitors, respectively.
CCN1 (CYR61) and CCN3 (NOV) signaling drives human trophoblast cells into senescence and stimulates migration properties.
Kipkeew Friederike,Kirsch Manuela,Klein Diana,Wuelling Manuela,Winterhager Elke,Gellhaus Alexandra
Cell adhesion & migration
During placental development, continuous invasion of trophoblasts into the maternal compartment depends on the support of proliferating extravillous trophoblasts (EVTs). Unlike tumor cells, EVTs escape from the cell cycle before invasion into the decidua and spiral arteries. This study focused on the regulation properties of glycosylated and non-glycosylated matricellular CCN1 and CCN3, primarily for proliferation control in the benign SGHPL-5 trophoblast cell line, which originates from the first-trimester placenta. Treating SGHPL-5 trophoblast cells with the glycosylated forms of recombinant CCN1 and CCN3 decreased cell proliferation by bringing about G0/G1 cell cycle arrest, which was accompanied by the upregulation of activated Notch-1 and its target gene p21. Interestingly, both CCN proteins increased senescence-associated β-galactosidase activity and the expression of the senescence marker p16. The migration capability of SGHPL-5 cells was mostly enhanced in response to CCN1 and CCN3, by the activation of FAK and Akt kinase but not by the activation of ERK1/2. In summary, both CCN proteins play a key role in regulating trophoblast cell differentiation by inducing senescence and enhancing migration properties. Reduced levels of CCN1 and CCN3, as found in early-onset preeclampsia, could contribute to a shift from invasive to proliferative EVTs and may explain their shallow invasion properties in this disease.
Dominant-negative inhibition of canonical Notch signaling in trophoblast cells does not disrupt placenta formation.
Shawber Carrie J,Brown-Grant Dex-Ann,Wu Tracy,Kitajewski Jan K,Douglas Nataki C
Proper development and function of the mammalian placenta requires interactions between embryo-derived trophoblasts and uterine endothelial cells to form mosaic vessels that facilitate blood flow to a developing conceptus. Notch signaling utilizes a cell-cell contact dependent mechanism to drive cell behaviors, such as differentiation and invasion. In mice, is needed for proper placentation and embryo survival. We used transgenic mice with a dominant-negative form of Mastermind-like1 and and drivers to inhibit canonical Notch signaling in trophoblasts. Both drivers resulted in robust placental expression of dominant-negative Mastermind-like1. All pregnancies progressed beyond mid-gestation and morphological analyses of placentas revealed no differences between mutants and controls. Our data suggest that mouse placentation occurs normally despite dominant negative inhibition of trophoblast canonical Notch signaling and that Notch2 signaling via the canonical pathway is not necessary for placentation.
Influence of miR-34a on preeclampsia through the Notch signaling pathway.
Liu J-J,Zhang L,Zhang F-F,Luan T,Yin Z-M,Rui C,Ding H-J
European review for medical and pharmacological sciences
OBJECTIVE:The aim of this study was to investigate the influence of micro-ribonucleic acid-34a (miR-34a) on preeclampsia through the Notch signaling pathway. PATIENTS AND METHODS:The expressions of miR-34a, Notch-1, Notch-2, and Notch-3 in the placenta of 39 preeclampsia patients and 42 normal patients were detected by immunohistochemistry and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The correlations between miR-34a expression with the expressions of Notch-1, Notch-2 and Notch-3 were analyzed, respectively. Besides, placental trophoblasts were isolated from preeclampsia patients and cultured in vitro. The expressions of miR-34a, Notch-1, Notch-2 and Notch-3 in placental trophoblasts were analyzed. Furthermore, the influences of miR-34a on the protein expressions of Notch-1, Notch-2, Notch-3, and hairy and enhancer of split-1 (Hes-1) in the Notch signaling pathway were analyzed by Luciferase reporter gene assay and Western blotting. The role of Notch in trophoblast invasion was investigated through the Notch inhibitors. In addition, its influence on the expression of urokinase-type plasminogen activator (uPA) was studied by miR-34a overexpression. RESULTS:The expressions of miR-34a and Notch-1 were correlated with preeclampsia in the placentas of preeclampsia patients and normal patients to a certain degree. The expression of miR-34a in preeclamptic placenta was significantly higher than that of the normal placenta (p<0.05). However, Notch-1 expression was markedly lower in preeclamptic placenta (p<0.05). No significant differences were found in the expressions of Notch-2 and Notch-3 between the two types of placentas (p>0.05). MiR-34a had a remarkable negative correlation with Notch-1 expression in the Notch family (p<0.001, r=-0.5775). RT-PCR results revealed that the mRNA expression of miR-34a in placental trophoblasts of patients with preeclampsia was notably higher than that of normal people (p<0.01). However, Western blotting demonstrated that the protein expressions of Notch-1, Notch-2 and Notch-3 exhibited the opposite results. Additionally, the protein expression of Notch-1, Notch-2, Notch-3 and Hes-1 in trophoblasts transfected with pre-miR-34a was significantly decreased. The treatment with Notch inhibitors markedly reduced the trophoblast invasion. Furthermore, miR-34a overexpression or intracellular domain of Notch (ICN) overexpression regulated uPA expression. CONCLUSIONS:MiR-34a regulates uPA system through the Notch signal transduction, thereby regulating the invasion of placental trophoblasts in patients with preeclampsia.
Notch signalling in placental development and gestational diseases.
Haider S,Pollheimer J,Knöfler M
Activation of Notch signalling upon cell-cell contact of neighbouring cells controls a plethora of cellular processes such as stem cell maintenance, cell lineage determination, cell proliferation, and survival. Accumulating evidence suggests that the pathway also critically regulates these events during placental development and differentiation. Herein, we summarize our present knowledge about Notch signalling in murine and human placentation and discuss its potential role in the pathophysiology of gestational disorders. Studies in mice suggest that Notch controls trophectoderm formation, decidualization, placental branching morphogenesis and endovascular trophoblast invasion. In humans, the particular signalling cascade promotes formation of the extravillous trophoblast lineage and regulates trophoblast proliferation, survival and differentiation. Expression patterns as well as functional analyses indicate distinct roles of Notch receptors in different trophoblast subtypes. Altered effects of Notch signalling have been detected in choriocarcinoma cells, consistent with its role in cancer development and progression. Moreover, deregulation of Notch signalling components were observed in pregnancy disorders such as preeclampsia and fetal growth restriction. In summary, Notch plays fundamental roles in different developmental processes of the placenta. Abnormal signalling through this pathway could contribute to the pathogenesis of gestational diseases with aberrant placentation and trophoblast function.
RNA interference-mediated knockdown of Notch-1 inhibits migration and invasion, down-regulates matrix metalloproteinases and suppresses NF-κB signaling pathway in trophoblast cells.
Yu Yang,Wang Leilei,Tang Weiwei,Zhang Dan,Shang Tao
Preeclampsia is well known to present with reduced trophoblast invasion into the placental bed. Notch-1, a ligand-activated transmembrane receptor, has been reported to be down-regulated in preeclamptic human placentas. This study was conducted to explore the role of Notch-1 in the cell migration and invasion of a human trophoblast cell line, JEG3 cells. Short hairpin RNA (shRNA)-mediated RNA interference was performed to effectively suppress the endogenous expression of Notch-1 at both mRNA and protein levels in JEG3 cells. Results of wound healing and transwell assays showed that knockdown of Notch-1 reduced trophoblast cell migration and invasion. The protein expressions and activities of matrix metalloproteinase (MMP)-2 and MMP-9 were reduced in JEG3 cell when Notch-1 was decreased. Furthermore, the epithelial-cadherin (E-cadherin) expression increased in JEG3 cells when Notch-1 was inhibited, whereas its suppressor Snail decreased in these cells. Moreover, knockdown of Notch-1 also suppressed NF-κB signaling pathway by inhibiting the phosphorylation of nuclear factor kappa B (NF-κB p65) inhibitor (IκBα) and the subsequent nuclear translocation of NF-κB subunit p65 in JEG3 cells. In summary, we demonstrate that Notch-1 contributes to trophoblast cell migration and invasion and that it may be involved in the pathology of human preeclampsia.
Notch-dependent RBPJκ inhibits proliferation of human cytotrophoblasts and their differentiation into extravillous trophoblasts.
Velicky P,Haider S,Otti G R,Fiala C,Pollheimer J,Knöfler M
Molecular human reproduction
Abnormal development of invasive trophoblasts has been implicated in the pathogenesis of human pregnancy diseases such as pre-eclampsia. However, critical signalling pathways controlling formation and differentiation of these cells have been poorly elucidated. Here, we provide evidence that the canonical Notch pathway, operating through Notch-dependent activation of its key regulatory transcription factor RBPJκ, controls proliferation and differentiation in villous explant cultures and primary trophoblasts of early pregnancy. Immunofluorescence of first trimester placental tissue revealed expression of RBPJκ and its co-activators, the MAML proteins, in nuclei of proliferative cell column trophoblasts (CCT) and differentiated, extravillous trophoblasts (EVTs). However, RBPJκ expression, transcript levels of the Notch target gene HES1 and activity of a Notch/RBPJκ-dependent luciferase reporter decreased during in vitro differentiation of primary cytotrophoblasts on fibronectin. Silencing of RBPJκ using silencing RNAs (siRNAs) increased proliferation of CCTs in floating villous explant cultures analysed by outgrowth and BrdU labelling. Similarly, down-regulation of the transcription factor enhanced BrdU incorporation in isolated primary cultures. However, motility of these cells was not affected. In addition, gene silencing of RBPJκ increased cyclin D1 expression in the two trophoblast model systems as well as markers of the differentiated, EVT, i.e. integrin α1, ADAM12 and T-cell factor 4. In summary, the data suggest that Notch-dependent RBPJκ activity could be required for balanced rates of trophoblast proliferation and differentiation in human placental anchoring villi preventing exaggerated trophoblast overgrowth as well as premature formation of EVTs.
Epidermal growth factor-like domain 7 promotes migration and invasion of human trophoblast cells through activation of MAPK, PI3K and NOTCH signaling pathways.
Massimiani M,Vecchione L,Piccirilli D,Spitalieri P,Amati F,Salvi S,Ferrazzani S,Stuhlmann H,Campagnolo L
Molecular human reproduction
Epidermal growth factor-like domain 7 (Egfl7) is a gene that encodes a partially secreted protein and whose expression is largely restricted to the endothelia. We recently reported that EGFL7 is also expressed by trophoblast cells in mouse and human placentas. Here, we investigated the molecular pathways that are regulated by EGFL7 in trophoblast cells. Stable EGFL7 overexpression in a Jeg3 human choriocarcinoma cell line resulted in significantly increased cell migration and invasiveness, while cell proliferation was unaffected. Analysis of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways showed that EGFL7 promotes Jeg3 cell motility by activating both pathways. We show that EGFL7 activates the epidermal growth factor receptor (EGFR) in Jeg3 cells, resulting in downstream activation of extracellular regulated kinases (ERKs). In addition, we provide evidence that EGFL7-triggered migration of Jeg3 cells involves activation of NOTCH signaling. EGFL7 and NOTCH1 are co-expressed in Jeg3 cells, and blocking of NOTCH activation abrogates enhanced migration of Jeg3 cells overexpressing EGFL7. We also demonstrate that signaling through EGFR and NOTCH converged to mediate EGFL7 effects. Reduction of endogenous EGFL7 expression in Jeg3 cells significantly decreased cell migration. We further confirmed that EGFL7 stimulates cell migration by using primary human first trimester trophoblast (PTB) cells overexpressing EGFL7. In conclusion, our data suggest that in trophoblast cells, EGFL7 regulates cell migration and invasion by activating multiple signaling pathways. Our results provide a possible explanation for the correlation between reduced expression of EGFL7 and inadequate trophoblast invasion observed in placentopathies.