Regulating Of CD82 At Human Maternal-Fetal Interface In The First-trimester Pregnancy

Implantation of human conceptus involves invasion of trophoblast cells into the uterine epithelium and the underlying stroma that undergoes a complex process of proliferation, migration and differentiation of embryo. A typical feature of placentation in humans is the trophoblasts with high degree invasion to gain access to the maternal circulation in the first trimester pregnancy. An impaired endovascular trophoblast invasion has been confirmed not only associated with pre-eclampsia, fetal intrauterine growth restriction, but also the first-trimester or late miscarriage.Trophoblast cells display a very unique capability, and physiologically invade into the surrounding tissue that is similar to tumour. Trophoblast and tumor cells share the same biochemical mediators:MMPs and TIMPs. However, as opposed to malignant invasion, trophoblastic invasion during implantation and placentation is stringently controlled both in space and time. The decidua forms a dense cellular matrix that is believed to generate a local cytokine milieu that promotes trophoblast attachment and acts as a physical barrier limiting trophoblast over-invasion.The CD82 gene (kangai1) encodes a 267 amino acid protein that contains four putative transmembrane domains. It is originally identified based on its function as a metastasis suppressor gene. CD82 plays an important role in inhibiting cancer cell motility, invasion and metastasis, and thus inhibits the formation of tumor metastases without affecting tumor growth. Gellersen et al have found that the expression of CD82 in decidual cell at human maternal-fetal interface is involved in decidual transformation from human endometrial stromal cells (ESCs). It is suggested that CD82 may be involved in the control of trophoblast cell invasion. Therefore, further investigation to elucidate the mechanism of CD82, and to find out how CD82 is regulated might help us to understand the regulation of embryonic implantation and trophoblastic invasion.In the present study, we focus on the possible molecular mechanism of CD82 on human trophoblast invasiveness in the early pregnancy, and roles of the CD82 in the cross-talking between trophoblast cells and DSCs, which contributes to trophoblast migration and ultimately to successful pregnancy. The identification of these pathways and molecule mechanisms could provide novel targets for the diagnosis and treatment of pathological pregnancies, enabling the translation of basic research discoveries into clinical applications.The expression of CD82 in tissues and cells was detected by semi-quantified RT-PCR, immunohistochemistry or immunocytochemical staining. We have demonstrated that the decidua and villi in the early pregnancy transcribe CD82 mRNA, but CD82 is only transcribed in primary DSCs, but not in primary trophoblast cells or BeWo cells (a choriocarcinoma cell line). The decidua as well as primary DSCs is stained positive of CD82 on the plasma membrane, but the villi, primary trophoblasts and BeWo cells are negative of CD82. The expression of CD82 in the decidua of normal early pregnancy and the unexplained miscarriage was detected by real-time PCR, immunohistochemistry and traditional western-blot, respectively. We have found that the mRNA level of CD82 in the decidua from the unexplained miscarriage is 97-fold higher than that of the normal early pregnancy (P<0.01). Consistent with transcription level, the decidua from the unexplained miscarriage has a much higher CD82 protein expression than that of the normal early pregnancy by immunohistochemistry and western blot (P<0.01), which suggests that the CD82 over-expression in decidua may restrict appropriate invasion of trophoblasts and lead to early pregnancy wastage.After isolation and culture of human trophoblast cells and DSCs from the first-trimester pregnancy, the primary DSCs were treated by the pregnancy-associated hormones, estrogen, progesterone and hCG, or proinflammatory cytokines, respectively. Then the CD82 expression of DSCs was determined by in-cell Western. We have found that hCG attenuates the expression of CD82, the maximal inhibition occurs at a concentration of lOkU/L (P<0.05), and progesterone can enhance this effect (P<0.05), but estrogen or progesterone alone cannot modulate the CD82 expression (P>0.05). These results suggest that the syncytiotrophoblast cells secrete hCG that probably participates in regulating the invasion in situ of extravillous trophoblast cells by down-regulating the expression of CD82 in DSCs. Our observation also demonstrates that LPS can promote the CD82 expression through stimulating the IL-1βsecretion.After isolation, purification and characterization of the first-trimester human trophoblast cells and DSCs, a model of direct and indirect co-culture was set up. The expression of CD82 in DSCs was interfered with siRNA, the BeWo cells were transfected with plasmid pcDNA3.1(+)-CD82, respectively. The effects of CD82 on invasion of trophoblast cells were investigated by Matrigel invasion assay, meanwhile, the invasion-related molecules in the transfected cells were detected by quantified real time RT-PCR, in-cell Western and immunofluorescence, respectively. We have found that the invasive index of human first-trimester trophoblast cells is significantly higher in co-culture with DSCs in CD82 silence than that of the si-negative control (P<0.01), and there is no difference between the direct and indirect co-culture (P>0.05), which suggests that the DSC-expressed CD82 can inhibit the invasion of human trophoblast cells by way of soluble molecules. Our observation also demonstrates that silencing of CD82 in DSCs significantly decreases the mRNA and protein levels of TIMP1 compared to the si-negative control (P<0.01 and P<0.05), and increase the transcription and protein levels of integrinβ1 and integrinαvβ3 (P<0.05). However, the mRNA and protein levels of MMP2, MMP9, TIMP2 and titin shows no statistical difference between the two groups (P>0.05). On the contrast, we have found that invasiveness of the CD82-expressed BeWo cells is significantly lower than that of the control (P<0.01). The TIMP1 mRNA and protein levels are increased, the integrinβ1 and integrinαvβ3 mRNA and protein levels are decreased after trophoblast cells are transfected with CD82 (P<0.05). Therefore, the CD82 inhibits the invasiveness of human trophoblasts in the first-trimester pregnancy through up-regulating the TIMP1 secretion. After isolation, purification from the first-trimester pregnancy, human DSCs were interfered for CD82, the BeWo cells were transfected with plasmid pcDNA3.1(+)-CD82, respectively. Then the key signal transduction molecules associated with invasion were detected by in-cell Western to screen the possible signal pathway. We have found that integrinβ1 and MAPK/ERK1/2 pathway might play an important role. Thereafter, we treated the CD82-silenced DSCs and CD82-expressed BeWo cells with U0126, MAPK/ERK pathway inhibitor, or anti-integrinβ1 neutralizing antibody, and then the invasiveness of the co-cultured trophoblast cells or BeWo cells was analyzed by Matrigel invasive assay, and the TIMP1 expression in the target cells was analyzed by in-cell Western. It has been found that CD82 in DSCs and BeWo cells significantly suppresses the invasion of human trophoblast cells and promotes protein expression of TIMP1. Compared to the si-negative control, the proportion of phospho-ERK1/2 to total ERK1/2 in the CD82-silenced DSCs was significantly increased (P<0.01). The proportion of phospho-ERK1/2 to total ERK1/2 in the DSCs is decreased after treated with anti-integrinβ1 neutralizing antibody (P<0.01), and anti-integrinβ1 neutralizing antibody can inhibit the up-regulating effect of CD82 silence on phosphorylation of ERK1/2 in DSCs. The inhibitor of MAPK/ERK1/2 signaling pathway, U0126 or anti-integrinβ1 neutralizing antibody can significantly abolish the invasiveness increase induced by CD82 silence. Thereafter, we investigated whether the decreased TIMP1 was involved in the up-regulation of CD82 silence on invasion of trophoblast cells by statistical relationship analysis. It has been also found that the expression of TIMP1 is in negative relation to the invasiveness of trophoblast cells. Hence, it is concluded that the CD82 in DSCs regulates the expression of TIMP1, participates in intercellular communication with human trophoblast cells, and then controls trophoblast invasion by inactivating integrinβ1/MAPK/MAPK3/1 signaling.After isolation and culture of human trophoblast cells and DSCs from the first-trimester pregnancy, the primary DSCs were treated with the supernatants derive of trophoblasts or/and recombinant human SDF-1, CCL2, anti-SDF-1 neutralizing antibody, anti-CXCR4 neutralizing antibody, anti-CCL2 neutralizing antibody or CCR2 antagonist, respectively. Then the CD82 expression in DSCs was determined by in-cell Western. We pre-treated trophoblasts and/or transfected DSCs with anti-CXCR4 neutralizing antibody, then constructed the co-culture of the two cells, and Matrigel invasion assay was used to detect the trophoblasts invasiveness in the co-culture. We have found that the supernatants from trophoblast cells can up-regulate the CD82 expression in DSCs (P<0.05), recombinant SDF-1 can promote the CD82 expression (P<0.05), and the CD82 expression in DSC is decreased after treated with anti-SDF-1 neutralizing antibody or anti-CXCR4 neutralizing antibody (P<0.05 or P<0.01). In the co-culture, the invasiveness of trophoblast cells was increased when DSCs were pre-treated with anti-CXCR4 neutralizing antibody (P<0.05). However, after trophoblast cells alone or in the co-culture were pre-treated with anti-CXCR4 neutralizing antibody, the trophoblast cells invasion was significantly decreased (P<0.05o r P<0.01).Therefore, it can be concluded that SDF-1 secreted by trophoblasts can not only promote the invasiveness of them in an autocrine manner, but also control the over-invasiveness of them through up-regulating the CD82 expression in DSCs in a paracrine manner.After isolation and culture of human trophoblast cells and DSCs from the first-trimester pregnancy, the primary DSCs were treated by CsA or the supernatants derived of the CsA-pretreated trophoblasts, and then treated with or without anti-SDF-1 neutralizing antibody or anti-CXCR4 neutralizing antibody, respectively. Thereafter, in-cell Western was used to evaluate the CD82 expression in DSCs. We constructed the trophoblasts, and DSCs respective culture or co-culture, and then detected the SDF-1 secretion in supernatants by ELISA assay. We also pre-treated trophoblasts and DSCs with anti-CXCR4 neutralizing antibody, then constructed the co-culture of the two cells, and then added by CsA. Matrigel invasion assay was used to detect the trophoblasts invasiveness. We have found that CsA cannot directly affect the KAI/CD82 expression in DSCs (P>0.05), but supernatants derived of the CsA pre-treated trophoblasts can further promote the CD82 expression in DSCs (P<0.01), and the anti-SDF-1 neutralizing antibody or anti-CXCR4 neutralizing antibody inhibits this effect (P<0.05 or P<0.01). CsA can stimulate the SDF-1 secretion in trophoblasts and co-culture unit (P<0.05 or P<0.01). When trophoblast cells alone or in the co-culture were pre-treated with anti-CXCR4 neutralizing antibody, the enhancement of trophoblast cells invasion induced by CsA was decreased (P<0.05 or P<0.01). Moreover, when DSCs were pre-treated with anti-CXCR4 neutralizing antibody, the increase of trophoblasts invasiveness induced by CsA was partly reversed (P<0.05). Therefore, CsA stimulates the SDF-1 secretion of trophoblasts, and enlarges the up-regulation of the trophoblast-derived SDF-1 on the KAI/CD82 expression in DSCs.In conclusion, CD82 has multiple modulating effects at the maternal-fetal interface:(1) CD82 promotes the TIMP1 expression and in turn suppresses the invasiveness of human first-trimester trophoblasts through inactivating integrinβ1MAPK/ERK1/2 signal pathway; (2) trophoblast cells enhances the CD82 expression in DSCs by secreting SDF-1 in a paracrine manner, and controls the over-invasion of trophoblast. Therefore, CD82 can promote the cross-talk between trophoblast cells and DSCs; (3) CsA stimulates the SDF-1 secretion of trophoblasts, and further up-regulates the CD82 expression in DSCs. Moreover, the expression of CD82 is also regulated by pregnancy-associated hormones hCG. Our results may elucidate human trophoblast invasion regulation mechanism, and provide a new idea for therapeutics of some pregnancy complications, such as miscarriage.