Human Blastocyst Secreted microRNA Regulate Endometrial Epithelial Cell Adhesion.
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ABSTRACT:
SUBMITTER: Cuman C
PROVIDER: S-EPMC4634783 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Trophinin is an intrinsic membrane protein expressed in trophectoderm cells of embryos and in uterine epithelial cells. Trophinin potentially mediates apical cell adhesion at human embryo implantation sites through trophinin-trophinin binding in these two cell types. Trophinin-mediated cell adhesion activates trophectoderm cells for invasion, whereas the effect of adhesion on maternal side is not known. We show that addition of GWRQ peptide, a previously established peptide that mimics trophinin-mediated cell adhesion, to human endometrial epithelial cells expressing trophinin induces their apoptosis. FAS involvement was excluded, as GWRQ did not bind to FAS, and FAS knockdown did not alter GWRQ-induced apoptosis. Immunoblotting analyses of protein kinases revealed an elevation of PKC-d protein in GWRQ-bound endometrial epithelial cells. In the absence of GWRQ, PKC-d associated with trophinin and remained cytoplasmic, but after GWRQ binding to the trophinin extracellular domain, PKC-d became tyrosine phosphorylated, dissociated from trophinin, and entered the nucleus. In PKC-d knockdown endometrial cells, GWRQ did not induce apoptosis.
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Project description:Successful embryo implantation requires a competent embryo, a receptive endometrium and synchronized communication between them. The selection of embryos with the highest implantation potential remains a challenge in the field of assisted reproductive technology. Moreover, little is known about the precise molecular mechanisms underlying embryo‑endometrium crosstalk. MicroRNAs (miRNAs/miRs) have been detected in the spent embryo culture medium (SCM); however, their functions at the preimplantation stage remain unclear. In the present study, human SCM samples were collected during in vitro fertilization/intracytoplasmic sperm injection‑embryo transfer and divided into implanted and not‑implanted groups according to the clinical pregnancy outcomes. Total RNA was extracted and six miRNAs (miR‑372‑3p, miR‑373‑3p, miR‑516b‑5p, miR‑517a‑3p, miR‑519d‑3p and miR‑520a‑3p) were selected for reverse transcription‑quantitative PCR (RT‑qPCR) analysis. The results revealed that miR‑372‑3p and miR‑519d‑3p were markedly increased in SCM from blastocysts that failed to implant compared with in blastocysts that implanted. The receiver operating characteristic curve analysis revealed that miR‑519d‑3p was superior to miR‑372‑3p in predicting pregnancy outcomes. In vitro miRNA uptake and cell adhesion assays were performed to determine whether miR‑519d‑3p could be taken up by endometrial epithelial cells and to examine the biological roles of miR‑519d‑3p after internalization. Potential targets of miR‑519d‑3p were verified using a dual‑luciferase reporter system. The results demonstrated that miR‑519d‑3p was taken up by human endometrial epithelial cells and that it may inhibit embryo adhesion by targeting HIF1α. Using RT‑qPCR, western blot analysis and flow cytometry assay, HIF1α was shown to inhibit the biosynthesis of fucosyltransferase 7 and sialyl‑Lewis X (sLex), a cell‑surface oligosaccharide that serves an important role in embryonic apposition and adhesion. In addition, a mouse model was established and the results suggested that miR‑519d‑3p overexpression hampered embryo implantation in vivo. Taken together, miRNAs in SCM may serve as novel biomarkers for embryo quality. Furthermore, miR‑519d‑3p was shown to mediate embryo‑endometrium crosstalk and to negatively regulate embryo implantation by targeting HIF1α/FUT7/sLex pathway.
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Project description:Wnt signaling is essential for the regulation of cell polarity and cell fate in the early embryogenesis of many animal species. Multiple Wnt genes and its pathway members are expressed in the mouse early embryo, raising the question whether they play any roles in preimplantation development. Dishevelled is an important transducer of divergent Wnt pathways. Here we show that three of the mouse Dishevelled proteins are not only expressed in oocytes and during preimplantation development, but also display distinct spatio-temporal localization. Interestingly, as embryos reach blastocyst stage, Dishevelled 2 becomes increasingly associated with cell membrane in trophectoderm cells, while at E4.5, Dishevelled 3 is highly enriched in the cytoplasm of ICM cells. These changes are coincident with an increase in the active form of beta-catenin, p120catenin transcription and decrease of Kaiso expression, indicating an upregulation of Wnt signaling activity before implantation. When Dishevelled-GFP fusion proteins are overexpressed in single blastomeres of the 4-cell stage embryo, the progeny of this cell show reduction in cell adhesiveness and a rounded shape at the blastocyst stage. This suggests that perturbing Dvl function interferes with cell-cell adhesion through the non-canonical Wnt pathway in blastocysts.
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Project description:Recurrent implantation failure (RIF) is a rather thorny problem in the clinical practice of assisted reproductive technology. Due to the complex aetiology of RIF, its pathogenesis is far from fully understood, and there is no effective treatment available. Here, We explored the regulatory mechanism of the four half-domains of LIM domain 1 (FHL1), which is significantly downregulated in the endometrium of RIF patients, in blastocyst-epithelial adhesion. Indeed, FHL1 expression was dramatically increased in normal female mid-secretory endometrial epithelial cells and was abnormally reduced in RIF patients. Furthermore, FHL1 overexpression promoted blastocyst-epithelial adhesion, and interfering with FHL1 expression in the mouse uterus significantly inhibited embryo implantation. Mechanistically, FHL1 did not regulate HOXA10 mRNA expression but increased HOXA10 protein stability and activated HOXA10, thereby promoting its regulation of downstream gene expression and the β3 integrin/FAK pathway. Meanwhile, FHL1 regulates HOXA10 function by increasing HOXA10 deacetylation through enhanced binding of HOXA10 and SIRT2. SIRT2-specific inhibitors can significantly inhibit this effect. In the endometrial epithelial cells of RIF patients, the correlation between FHL1 and HOXA10 and its downstream target genes has also been verified. Finally, our data indicated FHL1 is a regulatory molecule that promotes blastocyst-epithelial adhesion. Altogether, downstream dysfunction due to aberrant FHL1 expression is an important molecular basis for embryo implantation failure in patients with RIF and to provide new potential therapeutic targets.
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Project description:Flotillin-1 and flotillin-2 are two homologous, membrane raft associated proteins. Although it has been reported that flotillins are involved in cell adhesion processes and play a role during breast cancer progression, thus making them interesting future therapeutic targets, their precise function has not been well elucidated. The present study investigates the function of these proteins in cell-cell adhesion in non-malignant cells. We have used the non-malignant epithelial MCF10A cells to study the interaction network of flotillins within cell-cell adhesion complexes. RNA interference was used to examine the effect of flotillins on the structure of adherens junctions and on the association of core proteins, such as E-cadherin, with membrane rafts. We here show that the cadherin proteins of the adherens junction associate with flotillin-2 in MCF10A cells and in various human cell lines. In vitro, flotillin-1 and flotillin-2 directly interact with γ-catenin which is so far the only protein known to be present both in the adherens junction and the desmosome. Mapping of the interaction domain within the γ-catenin sequence identified the Armadillo domains 6-8, especially ARM domain 7, to be important for the association with flotillins. Furthermore, depletion of flotillins significantly influenced the morphology of the adherens junction in human epithelial MCF10A cells and altered the association of E-cadherin and γ-catenin with membrane rafts. Taken together, these observations suggest a functional role for flotillins, especially flotillin-2, in cell-cell adhesion in non-malignant epithelial cells.
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Project description:Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two ?-sites through a disintegrin and metalloprotease (ADAM) and at one ?-site through BACE1. Transmembrane cleavage by ?-secretase occurs at three ?-sites to generate extracellular A?-like fragments and at two ?-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of ?- and ?-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable.
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Project description:Integrin beta8 (ITGB8) is involved in the endometrial receptivity. The blastocyst first interacts with the luminal endometrial epithelial cells during its implantation; therefore, we have investigated the signaling of ITGB8 via FAK and VAV-RAC1 in the endometrial epithelial cells. Integrin beta8 was found elevated in epithelial cells at late-pre-receptive (day4, 1600 h) and receptive (day5, 0500 h) stages of endometrial receptivity period in the mouse. Integrins downstream molecule FAK has demonstrated an increased expression and phosphorylation (Y397) in the endometrium as well as in the isolated endometrial epithelial cells during receptive and post-receptive stages. Integrin beta8 can functionally interact with FAK, VAV and RAC1 as the levels of phosphorylated-FAK, and VAV along with the RAC-GTP form was reduced after ITGB8 knockdown in the endometrial epithelial cells and uterus. Further, VAV and RAC1 were seen poorly active in the absence of FAK activity, suggesting a crosstalk of ITGB8 and FAK for VAV and RAC1 activation in the endometrial epithelial cells. Silencing of ITGB8 expression and inhibition of FAK activity in the Ishikawa cells rendered poor attachment of JAr spheroids. In conclusion, ITGB8 activates VAV-RAC1 signaling axis via FAK to facilitate the endometrial epithelial cell receptivity for the attachment of blastocyst.
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Project description:BackgroundThe human endometrium is a highly regenerative tissue that is believed to have two main types of stem cells: endometrial mesenchymal/stromal stem cells (eMSCs) and endometrial epithelial stem cells (eESCs). So far, eMSCs have been extensively studied, whereas the studies of eESCs are constrained by the inability to culture and expand them in vitro. The aim of this study is to establish an efficient method for the production of eESCs from human endometrium for potential clinical application in intrauterine adhesion (IUA).ResultsHere we developed a culture condition with a combination of some small molecules for in vitro culturing and expansion of human SSEA-1+ cells. The SSEA-1+ cells exhibited stem/progenitor cell activity in vitro, including clonogenicity and differentiation capacity into endometrial epithelial cell-like cells. In addition, the SSEA-1+ cells, embedded in extracellular matrix, swiftly self-organized into organoid structures with long-term expansion capacity and histological phenotype of the human endometrial epithelium. Specifically, we found that the SSEA-1+ cells showed stronger therapeutic potential than eMSCs for IUA in vitro. In a rat model of IUA, in situ injection of the SSEA-1+ cells-laden chitosan could efficiently reduce fibrosis and facilitate endometrial regeneration.ConclusionsOur work demonstrates an approach for isolation and expansion of human eESCs in vitro, and an appropriate marker, SSEA-1, to identify eESCs. Furthermore, the SSEA-1+ cells-laden chitosan might provide a novel cell-based approach for IUA treatment. These findings will advance the understanding of pathophysiology during endometrial restoration which may ultimately lead to more rational clinical practice.
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Project description:Cell adhesion molecule 1 (CADM1), expressed by human lung mast cells (HLMCs), mediates their adhesion to airway smooth muscle (ASM), and contributes to ASM-dependent HLMC proliferation and survival. CADM1 is expressed in alternatively spliced isoforms, but those present in HLMCs and their function are not known. We cloned three functional and one cryptic non-functional isoform with alternative splicing between exons 7/11 and 1/2, respectively, from HLMCs and human MC lines (HMC-1 and LAD2). Differentiated HLMCs and LAD2 cells expressed the functional isoform SP4 containing exons 7/8/11 (~80% of clones), as well as SP1 (exons 7/8/9/11) and a novel SP6 (exons 7/8/9/10/11). In contrast, immature HMC-1 cells expressed only functional SP4. SP4 overexpression in HMC-1 cells and HLMCs augmented homotypic adhesion to a greater extent than SP1 in various conditions. In contrast, CADM1 downregulation abolished homotypic adhesion, indicating that CADM1 is the sole receptor mediating mast cell aggregation. CADM1-mediated adhesion was enhanced by the presence of cell survival factors. SP1 overexpression in HMC-1 cells compromised survival compared to SP4 overexpression or control. CADM1 downregulation resulted in reduced viability and decreased expression of the pro-survival protein Mcl-1(L), but not Blc-2 or Bcl-X(L), and increased caspase-3/7 activity in both HMC-1 cells and HLMCs. This coincided with decreased basal Kit levels in HLMCs. In summary, human MCs express multiple CADM1 isoforms which exhibit differential regulation of survival and homotypic adhesion. The most highly expressed SP4 isoform is likely to contribute to MC aggregation and longevity in mastocytosis, and augment the pathophysiology of allergic diseases.
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