The Role Of Human Leukocyte Antigen G5in Modulating Trophoblast Invasion And Endometrial Receptivity

BackgroundHLA-G is a non-classical class Ib HLA molecule which was first isolated fromhuman extra-villous trophoblastic (EVT) cells. Compared with other isoforms, HLA-G5isthe―full-length‖soluble isoform which plays the most important role in implantation.HLA-G5is a modulator of fetal-maternal interactions. It promotes endometrial receptivityof the blastocyst; it controls placenta growth and development; and it helps to maintain alocal immunosuppressive environment. Intensively studies have been well identified therole of HLA-G5in modulating immune tolerance during pregnancy. However, whetherHLA-G5exerts a direct effect on trophoblast or endometrium function remains unclear. Wehypothesized that HLA-G5directly modulating the function of the endometrial cells andtrophoblasts partly through binding to its specific receptors on these cells to activatedownstream signaling pathway.ObjectiveThere were three objectives in this study:1. To produce and purify HLA-G5recombinant protein;2. To study the biological effect of soluble HLA-G5on trophoblastic and endometrialcells;3. To study the receptors and mechanisms those mediate the biological effect ofHLA-G5.Methods1. PCR, Western blot and immunostaining were used to analyze the expression ofHLA-G5proteins and its receptors KIR2DL4and LILRB1in trophoblastic andendometrial cells; antibodies against KIR2DL4and LILRB1were used in the binding assay and invasion assay to confirm the presence and function of thereceptors;2. HLA-G5cDNA was cloned into pET-15b and transformed into E. coli BL21;HLA-G5protein was over-expressed under the induction of0.5mMisopropy-β-D-thiogalactoside at30°C for3hours and the recombinant protein waspurified and refolded by dialysis;3. Flow cytometry was used to assess the binding of florescence-labeled HLA-G5recombinant protein to trophoblastic cells and endometrial cells;4. The fluorometric CyQUANT NF Cell proliferation assay was used to study theproliferation of the JAr and JEG-3cells;5. Cell Biolabs CytoSelect96-well Cell Migration Assay was used for studyingtrophoblast migration;6. Matrigel pre-coated invasion chamber was used to measure the invasiveness oftrophoblasts;7. ELISA and Cytokine Array were used to measure the levels of cytokines in theculture media of trophoblastic and endometrial cells;8. Quantitative polymerase chain reaction (Q-PCR) was used to measure theexpression of proteinases uPA and MMPs; gelatin zymography was used todetermine the activity of secreted proteinases MMP-2and-9; QIA125UrokinaseActivity Assay kit was used to measure the activity of proteinase uPA;9. Western blot was used to study the signaling pathways involved in trophoblastinvasion; ERK/SAPK inhibitors were used to confirm the effect of MAPKspathway in trophoblast invasion;10. An optimized model of Jar spheroids and Ishikawa monolayer co-culture modelwas employed to study the effect of HLA-G5in the embryo-endometrialepithelium attachment;11. Western blot was used to study the changes of adhesion molecules (E-cadherin and β-catenin) in endometrial cells after HLA-G5treatment;12. The untreated groups were used as controls in this study; statistical analysis wasdone by using ONE WAY ANOVA or t-test for normally distributed data andnon-parametric tests for skewed data with Sigma Plot11.0.Results1. HLA-G5recombinant protein was over-expressed, purified and refolded; theproduct was confirmed by Western blotting and by the mass spectrometry;2. The presence of HLA-G5was confirmed in JEG-3cells; the presence of HLA-G5receptors KIR2DL4and LILRB1were confirmed in JAr, JEG-3and Ishikawacells;3. HLA-G5bound to JAr and JEG-3cells via specific receptors;4. HLA-G5increased the invasion but not migration of trophoblasts;5. HLA-G5altered mRNA transcription and activity of uPA and MMPs;6. The levels of IL-6in the media of JAr and JEG-3cells were not affected byHLA-G5; but IL-6in the medium of Ishikawa cells was decreased after HLA-G5treatment;7. Receptor KIR2DL4and LILRB1mediate the HLA-G5induced trophoblastinvasion;8. HLA-G5induced phosphorylation of ERKs and SAPK/JNKs in trophoblasts;9. HLA-G5increased the attachment rate of JAr spheroids on Ishikawa monolayer;10. HLA-G5increased the expression of adhesion molecules (E-cadherin and β-catenin) in endometrial cells.Conclusions1. HLA-G5prokaryotic cell expression system was successfully established toproduce high-quantity and high-purity HLA-G5recombinant protein;2. HLA-G5induced trophoblast invasion by increasing uPA and MMPsexpression/activity, which was mediated by HLA-G5receptors KIR2DL4and LILRB1via the MAPKs signaling pathway;3. HLA-G5induced the attachment of JAr spheroid on Ishikawa monolayer, whichmay be mediated by KIR2DL4and LILRB1. Decrease of IL-6may be responsiblefor the weakened allo-response to exogenous antigens, while suppression ofE-cadherin and active-beta-catenin may contribute to weakening of endometrialepithelia cell-cell contact for trophoblast attachment.