Functional embryo–maternal interactions occur during the embryo implantation and placentation. Extracellular vesicles with microRNA (miR) between cells have been considered of critical importance for embryo implantation and the programming of human pregnancy. MiR-138-5p functions as the transcriptional regulator of G protein-coupled receptor 124 (GPR124). However, the signaling pathway of miR138-5p- and GPR124-adjusted NLRP3 inflammasome activation remains unclear. In this study, we examine the roles of the miR138-5p and GPR124-regulated inflammasome in embryo implantation and early pregnancy. Human decidual stromal cells were isolated from the abortus tissue and collected by curettage from missed abortion patients and normal pregnant women at 6– to 12-week gestation, after informed consent. Isolated extracellular vesicles from decidua and decidual stromal cells were confirmed by transmission electron microscopy (TEM). Next-Generation Sequencing (NGS) and microarray were performed for miR analysis. The predicated target genes of the differentially expressed miR were analyzed to identify the target genes and their pathway. We demonstrated the down-regulation of miR-138-5p and the overexpression of GPR124 in spontaneous miscarriage compared to normal pregnancy. We also showed the excessive activation of the NLRP3 inflammasome in spontaneous miscarriage compared to normal pregnancy. Here, we newly demonstrate that the miR-138-5p and GPR124-adjusted NLRP3 inflammasome were expressed in extracellular vesicles derived from decidua and decidual stromal cells, indicating that the miR-138-5p, GPR124 and NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome have a potential modulatory role on the decidual programming and placentation of human pregnancy. Our findings represent a new concept regarding the role of extracellular vesicles, miR-138-5p, GPR124, and the NLRP3 inflammasome in normal early pregnancy and spontaneous miscarriage.
Background In endothelial cells, phospholipase C (PLC) β1-activated Ca2+ is a crucial second messenger for the signaling pathways governing angiogenesis. PLCβ1 is inactivated by complexing with an intracellular protein called translin-associated factor X (TRAX). This study demonstrates specific interactions between Globo H ceramide (GHCer) and TRAX, which highlight a new angiogenic control through PLCβ1 activation. Methods Globo-series glycosphingolipids (GSLs), including GHCer and stage-specific embryonic antigen-3 ceramide (SSEA3Cer), were analyzed using enzyme-linked immunosorbent assay (ELISA) and Biacore for their binding with TRAX. Angiogenic activities of GSLs in human umbilical vein endothelial cells (HUVECs) were evaluated. Molecular dynamics (MD) simulation was used to study conformations of GSLs and their molecular interactions with TRAX. Fluorescence resonance energy transfer (FRET) analysis of HUVECs by confocal microscopy was used to validate the release of PLCβ1 from TRAX. Furthermore, the in vivo angiogenic activity of extracellular vesicles (EVs) containing GHCer was confirmed using subcutaneous Matrigel plug assay in mice. Results The results of ELISA and Biacore analysis showed a stable complex between recombinant TRAX and synthetic GHCer with Kd of 40.9 nM. In contrast, SSEA3Cer lacking a fucose residue of GHCer at the terminal showed ~ 1000-fold decrease in the binding affinity. These results were consistent with their angiogenic activities in HUVECs. The MD simulation indicated that TRAX interacted with the glycan moiety of GHCer at amino acid Q223, Q219, L142, S141, and E216. At equilibrium the stable complex maintained 4.6 ± 1.3 H-bonds. TRAX containing double mutations with Q223A and Q219A lost its ability to interact with GHCer in both MD simulation and Biacore assays. Removal of the terminal fucose from GHCer to become SSEA3Cer resulted in decreased H-bonding to 1.2 ± 1.0 by the MD simulation. Such specific H-bonding was due to the conformational alteration in the whole glycan which was affected by the presence or absence of the fucose moiety. In addition, ELISA, Biacore, and in-cell FRET assays confirmed the competition between GHCer and PLCβ1 for binding to TRAX. Furthermore, the Matrigel plug assay showed robust vessel formation in the plug containing tumor-secreted EVs or synthetic GHCer, but not in the plug with SSEA3Cer. The FRET analysis also indicated the disruption of colocalization of TRAX and PLCβ1 in cells by GHCer derived from EVs. Conclusions Overall, the fucose residue in GHCer dictated the glycan conformation for its complexing with TRAX to release TRAX-sequestered PLCβ1, leading to Ca2+ mobilization in endothelial cells and enhancing angiogenesis in tumor microenvironments.
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