miR-27b-5p inhibits BeWo cells fusion by regulating WNT2B and enzyme involved in progesterone synthesis.

PROBLEM: The miRNAs show placenta-specific expression patterns, which alter during pregnancy-related complications. In present study, the role of miR-27b-5p during forskolin-mediated BeWo cells fusion has been investigated. METHOD OF STUDY: The fusion of BeWo cells in response to forskolin treatment (25 µM) was studied by desmoplakin I+II staining. Expression profile of miR-27b-5p by qRT-PCR and its targets HSD3ß1 and WNT2B by qRT-PCR and in Western blot were studied. The effect of overexpression of miR-27b-5p and silencing of HSD3ß1 & WNT2B by siRNA on forskolin-mediated BeWo cells fusion and secretion of hCG and progesterone by ELISA was investigated. RESULTS: Time-dependent down-regulation in the expression of miR-27b-5p in forskolin-treated BeWo cells has been confirmed by qRT-PCR. Overexpression of miR-27b-5p significantly inhibits forskolin-mediated BeWo cells fusion as well as hCG & progesterone secretion. HSD3ß1 and WNT2B were identified as targets of miR-27b-5p and are up-regulated in forskolin-treated BeWo cells. Overexpression of miR-27b-5p in BeWo cells downregulates their expression. Further, luciferase reporter assay revealed that miR-27b-5p directly target expression of both HSD3ß1 and WNT2B. Silencing of both HSD3ß1 and WNT2B leads to a significant reduction in forskolin-mediated BeWo cells fusion with concomitant decrease in the secretion of progesterone or/and hCG. Decrease in forskolin-mediated cells fusion observed in miR-27b-5p mimic transfected BeWo cells could be rescued by the overexpression of both HSD3ß1 and WNT2B. CONCLUSION: These observations suggest that reduced miR-27b-5p in forskolin-treated BeWo cells leads to increased secretion of progesterone and hCG due to loss of repressional control on HSD3ß1 and WNT2B.

HGF promotes HTR-8/SVneo cell migration through activation of MAPK/PKA signaling leading to up-regulation of WNT ligands and integrins that target ß-catenin.

Inadequate migration and invasion of the trophoblast cells during embryo implantation is one of the reasons for pregnancy-related complications such as intrauterine growth restriction and preeclampsia. In the present study, relevance of WNT ligands and integrins associated with hepatocyte growth factor (HGF)-mediated migration of HTR-8/SVneo trophoblastic cells has been investigated. Treatment of HTR-8/SVneo cells with HGF led to a dose-dependent increase in their migration. RT-PCR studies revealed a significant increase in the transcripts of WNT4, WNT11, ITGA2, and ITGAV, which was further confirmed at protein level by Western blotting. HGF treatment also led to increased expression of integrin α2ß1 and αVß5 in HTR-8/SVneo cells. Silencing of WNT4, WNT11, ITGA2, and ITGAV by siRNA led to a significant decrease in HGF-mediated migration of cells. Treatment of cells with HGF led to activation of mitogen-activated protein kinases (MAPK) and protein kinase A (PKA) signaling pathways. Inhibition of MAPK/PKA, by selective inhibitors, led to decrease in the expression of above WNT ligands and integrins. Silencing of WNT4/WNT11 led to concomitant decrease in the expression of ITGA2 and ITGAV and vice versa. HGF treatment also led to significant increase in ß-catenin expression, a downstream target of both WNT ligands and integrins. Silencing of ß-catenin led to decrease in HGF-mediated migration. ß-catenin expression was also down-regulated in WNT4/WNT11/ITGA2/ITGAV silenced cells suggesting a possible cross-communication of WNT ligands and integrins via ß-catenin. These studies have established the significance of WNT4/WNT11 as well as ITGA2/ITGAV during HGF-mediated migration of HTR-8/SVneo trophoblastic cells.

Role of STAT signaling and autocrine action of chemokines during H2 O 2 induced HTR-8/SVneo trophoblastic cells invasion.

During pregnancy, regulated generation of reactive oxygen species (ROS) is important for activation of signaling pathways and placentation. In the current study, the effect of H2 O2 on invasion of HTR-8/SVneo cells, a human extravillous trophoblast cell line, is investigated. Treatment of HTR-8/SVneo cells for 24 hr with H 2 O2 (25 µM) leads to a significant increase in invasion without affecting cell proliferation, viability, and apoptosis. Concomitantly, a significant increase in the matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinases-1 (TIMP-1) ratio is observed. Further, significant increase in phosphorylation of signal transducer and activator of transcription 1 (STAT-1) and STAT-3 (both at ser727 residue) is observed on treating HTR-8/SVneo cells with 25 µM of H2 O2 accompanied by an increase in the secretion of interleukin-8 (IL-8) and macrophage inflammatory protein-1ß (MIP-1ß). A significant decrease in H2 O2 -mediated invasion of HTR-8/SVneo cells and reduced expression of IL-8 and MIP-1ß accompanied by decrease in MMP-9/TIMP-1 ratio are observed on inhibiting STAT-1 and STAT-3 by small interfering RNA (siRNA). Inhibition of STAT-1 activity by fludarabine and STAT-3 activity by Stattic also leads to a decrease in H2 O2 -mediated increase in HTR-8/SVneo cell invasion. Inhibition of IL-8 and MIP-1ß by siRNA also leads to a significant decrease in both basal and H2 O2 -mediated invasion. Interestingly, inhibition of MIP-1ß by siRNA leads to a significant reduction in H2 O2 -mediated increase in IL-8. However, no significant effect of IL-8 silencing on H2 O2 -mediated MIP-1ß expression was observed. From the above results, it can be concluded that H2 O2 activates STAT signaling, MIP-1ß & IL-8 secretion and increases MMP-9/TIMP-1 ratio leading to an increased invasion of HTR-8/SVneo cells without affecting their viability.

Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation.

Nur-77, a member of the NR4A sub-family of nuclear orphan receptors, is downregulated in the placentae of pre-eclamptic women. Here, we investigate the relevance of Nor-1, Nurr-1 and Nur-77 in trophoblastic cell differentiation. Their transcript levels were found to be significantly upregulated in BeWo cells treated with forskolin. The maximum increase was observed after 2 h, with a second peak in the expression levels after 48 h. The expression of NR4A sub-family members was also found to be upregulated in BeWo cells after treatment with hCG and GnRH. A similar significant increase was observed at the respective protein levels after 2 and 48 h of treatment with forskolin, hCG or GnRH. Silencing Nor-1, Nurr-1 or Nur-77 individually did not show any effect on forskolin-, hCG- and/or GnRH-mediated BeWo cell fusion and/or hCG secretion. After silencing any one member of the NR4A sub-family, an increase in the transcript levels of the other sub-family members was observed, indicating a compensatory effect due to their functional redundancy. Simultaneously silencing all three NR4A sub-family members significantly downregulated forskolin- and hCG-mediated BeWo cell fusion and/or hCG secretion. However, a considerable amount of cell death occurred after forskolin or hCG treatment as compared to the control siRNA-transfected cells. These results suggest that the NR4A sub-family of nuclear orphan receptors has a role in trophoblastic cell differentiation.

Relevance of Wnt10b and activation of ß-catenin/GCMa/syncytin-1 pathway in BeWo cell fusion.

PROBLEM: To study the involvement of specific Wnt(s) ligand during trophoblastic BeWo cell differentiation. METHOD OF STUDY: BeWo cells on treatment with forskolin/human chorionic gonadotropin (hCG) were studied for cell fusion by desmoplakin I+II staining and/or hCG secretion by ELISA. Levels of Wnt10b/ß-catenin/glial cell missing a (GCMa)/syncytin-1 were studied by qPCR/Western blotting in forskolin-/hCG-treated control siRNA and Wnt10b silenced BeWo cells. RESULTS: BeWo cells on treatment with hCG (5 IU/mL) led to a 94-fold increase in Wnt10b transcript. Wnt10b silencing showed significant decrease in forskolin-/hCG-mediated BeWo cell fusion and/or hCG secretion. It led to down-regulation of ß-catenin (nuclear and cytoplasmic), GCMa and syncytin-1 expression. Treatment of BeWo cells with H89, protein kinase A (PKA) signaling inhibitor, significantly reduced forskolin-/hCG-induced Wnt10b, ß-catenin, and syncytin-1 expression, which also resulted in reduced cell fusion. CONCLUSION: Wnt10b is involved in forskolin/hCG-mediated BeWo cell fusion via ß-catenin/GCMa/syncytin pathway, which may also involve activation of PKA.

Cell Signaling Pathways Involved During Invasion and Syncytialization of Trophoblast Cells.

Implantation involves an extensive cross talk between the trophoblast cells and the receptive endometrium through embryonic as well as endometrial-derived factors that regulate the invasion and migration of trophoblast cells and also syncytia formation. Any aberration in this highly regulated process may lead to pregnancy complications such as preeclampsia, intrauterine growth restriction, or even pregnancy failure. How various cytokines and growth factors act by activating various cell signaling pathways leading to the expression of the effector molecules have been reviewed, which control invasion and migration of trophoblast cells and syncytialization. The gaps in our current understanding of the various signaling pathways, activated by different cytokines/growth factors, their possible cross talk for optimized effector function(s), and future prospects in this field have been discussed.

Alpha or beta human chorionic gonadotropin knockdown decrease BeWo cell fusion by down-regulating PKA and CREB activation.

The aim of the present study is to delineate the role of human chorionic gonadotropin (hCG) in trophoblast fusion. In this direction, using shRNA lentiviral particles, α- and ß-hCG silenced 'BeWo' cell lines were generated. Treatment of both α- and ß-hCG silenced BeWo cells with either forskolin or exogenous hCG showed a significant reduction in cell fusion as compared with control shRNA treated cells. Studies by qRT-PCR, Western blotting and immunofluorescence revealed down-regulation of fusion-associated proteins such as syncytin-1 and syndecan-1 in the α- and ß-hCG silenced cells. Delineation of downstream signaling pathways revealed that phosphorylation of PKA and CREB were compromised in the silenced cells whereas, no significant changes in p38MAPK and ERK1/2 phosphorylation were observed. Moreover, ß-catenin activation was unaffected by either α- or ß-hCG silencing. Further, inhibition of PKA by H89 inhibitor led to a significant decrease in BeWo cell fusion but had no effect on ß-catenin activation suggesting the absence of non-canonical ß-catenin stabilization via PKA. Interestingly, canonical activation of ß-catenin was associated with the up-regulation of Wnt 10b expression. In summary, this study establishes the significance of hCG in the fusion of trophoblastic BeWo cells, but there may be additional factors involved in this process.

LIF-STAT signaling and trophoblast biology.

Leukemia inhibitory factor (LIF) is a pleiotropic growth factor that regulates several biological functions. This review focuses on the LIF-dependent STAT activation and its impact on modulation of trophoblast functions during embryo implantation. LIF is mainly produced by the maternal endometrium at the time of implantation while its receptors are present both on the endometrium and trophoblasts. It might influence blastocyst attachment through STAT3 activation and expression of integrins. After attachment of the blastocyst, trophoblasts undergo proliferation and differentiation into invasive EVTs and non-invasive STBs. Under in vitro conditions, LIF regulates all these processes through activation of STAT- and MAPK-dependent signaling pathways. The observations that LIF and STAT3 knockout mice are infertile further strengthen the notion about the critical involvement of LIF-mediated signaling during embryo implantation. Hence, a better understanding of LIF-STAT signaling would help in improving fertility as use of LIF in in vitro blastocyst culture improves the implanting ability of blastocyst after IVF.

Mammalian zona pellucida glycoproteins: structure and function during fertilization.

Zona pellucida (ZP) is a glycoproteinaceous translucent matrix that surrounds the mammalian oocyte and plays a critical role in the accomplishment of fertilization. In humans, it is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4, whereas mouse ZP is composed of ZP1, ZP2 and ZP3 (Zp4 being a pseudogene). In addition to a variable sequence identity of a given zona protein among various species, human ZP1 and ZP4 are paralogs and mature polypeptide chains share an identity of 47%. Employing either affinity purified native or recombinant human zona proteins, it has been demonstrated that ZP1, ZP3 and ZP4 bind to the capacitated human spermatozoa and induce an acrosome reaction, whereas in mice, ZP3 acts as the putative primary sperm receptor. Human ZP2 only binds to acrosome-reacted spermatozoa and thus may be acting as a secondary sperm receptor. In contrast to O-linked glycans of ZP3 in mice, N-linked glycans of human ZP3 and ZP4 are more relevant for induction of the acrosome reaction. Recent studies suggest that Sialyl-Lewis(x) sequence present on both N- and O-glycans of human ZP play an important role in human sperm-egg binding. There are subtle differences in the downstream signaling events associated with ZP3 versus ZP1/ZP4-mediated induction of the acrosome reaction. For example, ZP3 but not ZP1/ZP4-mediated induction of the acrosome reaction is dependent on the activation of the Gi protein-coupled receptor. Thus, various studies suggest that, in contrast to mice, in humans more than one zona protein binds to spermatozoa and induces an acrosome reaction.