Chuan-Ling Tang

CD82 gene suppression in endometrial stromal cells leads to increase of the cell invasiveness in the endometriotic milieu

Tetraspanin CD82 is a wide-spectrum tumor metastasis suppressor that inhibits motility and invasiveness of cancer cells. Endometriosis is a benign gynecological disorder, but appears malignant behaviors including invasion, ectopic implantation and recurrence. This study is to elucidate the role of CD82 expression regulation in the pathogenesis of endometriosis. The short interfering RNA silence was established to analyze the roles of CD82, chemokine CCL2, and its receptor CCR2 in the invasiveness of endometrial stromal cells (ESCs). We have found that the mRNA and protein levels of CD82 in the primary normal ESCs from endometrium without endometriosis are significantly higher than that of the primary ESCs from eutopic endometrium and ectopic tissue. CD82 inhibits the invasiveness of ESCs by downregulating CCL2 secretion and CCR2 expression via mitogen-activated protein kinase (MAPK) and integrinβ1 signal pathway, and in turn upregulating the expression of TIMP1 and TIMP2 in an autocrine manner. The combination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with 17β-estradiol can promote the invasion of ESCs via suppressing CD82 expression and stimulating CCL2 secretion and CCR2 expression, and the enhanced interaction of CCL2-CCR2 recruits more macrophages into the ectopic milieu in a paracrine manner, which further downregulates CD82 expression in the ectopic ESCs. Our study has demonstrated for the first time that the abnormal lower CD82 expression in ESCs induced by TCDD and estrogen may be an important molecular basis of endometriosis pathogenesis through enhancing the CCL2 secretion and CCR2 expression and the invasion of ESCs via MAPK and integrinβ1 signal pathway.

The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway in Human First-Trimester Pregnancy

CD82 is recognized as a wide-spectrum tumor metastasis suppressor that inhibits cancer cell motility and invasiveness. At the human maternal-fetal interface, the decidua is believed to effectively limit the inappropriate invasion of trophoblasts. Here we have found the transcription and translation of CD82 in decidual stromal cells (DSCs), whereas trophoblast cells do not express CD82. The in-cell Western analysis reveals attenuation of CD82 translation in DSCs by human chorionic gonadotropin (hCG), but not by estrogen or progesterone. It is demonstrated that silencing of CD82 by RNA interference increases integrinβ1, decreases TIMP1 expression in DSCs, and promotes the invasion of the first-trimester human trophoblasts in the coculture. Moreover, U0126, or anti-integrinβ1 neutralizing antibody, reverses the decreased TIMP1 expression and the increased invasiveness of trophoblast cells, and the antibody also inhibits the MAPK3/1 phosphorylation induced by CD82 silence. After transfection with CD82, the invasive index of BeWo cells decreases significantly with TIMP1 increase. The results above indicate that the DSCs-expressed CD82 up-regulates the expression of TIMP1 in an autocrine manner and inhibits the invasiveness of human first-trimester trophoblast cells partly through the integrinβ1/MAPK/MAPK3/1 signaling pathway. Furthermore, we have found that the mRNA and protein level of CD82 in decidua of the miscarriage is significantly higher than that of the normal early pregnancy, which implies that the abnormal higher CD82 expression in decidua restricts appropriate invasion of trophoblasts that leads to early pregnancy wastage.

The CXCL12/CXCR4 axis is involved in the maintenance of Th2 bias at the maternal/fetal interface in early human pregnancy

The regulatory mechanism of Th2 bias at the maternal/fetal interface remains unclear. In this study, we characterized cytokine production in decidual stromal cells (DSCs), decidual immune cells (DICs) and embryo-derived trophoblast cells, and investigated the regulation of CXCL12/CXCR4 interaction on Th2 bias at the maternal/fetal interface in early human pregnancy. We found differential production of Th1-type and Th2-type cytokines by trophoblasts, DSCs and DICs. The secretion of these cytokines varied in different cell cocultures, conduced to Th2 bias. Flow cytometry showed that coculture of trophoblasts with DSCs and DICs significantly increased IL-4 and IL-10 production in trophoblasts, and IL-10 production in DSCs. However, the coculture of trophoblasts with DSCs and DICs significantly increased interferon (IFN)-γ expression in DSCs, and tumor-necrosis factor (TNF)-α expression in DICs. No change was seen in Th1-type cytokine production in trophoblasts, and in Th2-type cytokine production in DICs in all cocultures. Furthermore, pre-treatment with anti-CXCR4 neutralizing antibody upregulated the production of the Th1-type cytokines IFN-γ and TNF-α, and downregulated the production of the Th2-type cytokines IL-4 and IL-10, in trophoblasts, DSCs, DICs or their cocultures. Interestingly, rhCXCL12 inhibited production of the Th1-type cytokine TNF-α and enhanced the expression of the Th2-type cytokines such as IL-4 and IL-10 in DICs; this effect was abrogated by anti-CXCR4 antibody. Our present study has elucidated the individual contributions of component cells to the shaping of Th2 bias, and uncovered a complicated cross-talk via the CXCL12/CXCR4 signal at the maternal/fetal interface in early human pregnancy.

CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells

Introduction Our previous study has demonstrated Cyclosporin A (CsA) promotes the proliferation of human trophoblast cells. Therefore, we further investigate the intracellular signaling pathway involved in the CsA-induced proliferation of human trophoblast cells. Methods Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the regulation of CsA on CXCL12 secretion in human trophoblast cells. Immunofluorescence analysis and western blotting analysis were used to investigate the role of CXCL12/CXCR4 axis in the CsA-induced epidermal growth factor receptor (EGFR) phosphorylation in human trophoblast cells. 5-bromo-2′-deoxyuridine (BrdU) cell proliferation assay was performed to analyze the involvement of EGFR and its downstream extracellular signal-regulated protein kinase (ERK) signaling pathway in the CsA-induced proliferation of human trophoblast cells. Results Low concentration of CsA promoted the secretion of CXCL12, and recombinant human CXCL12 promoted the phosphorylation of EGFR in primary human trophoblast cells and choriocarcinoma cell line JEG-3. The inhibition of CXCL12 or CXCR4 by either neutralizing antibodies or small interfering RNA (siRNA) could completely block the CsA-induced EGFR phosphorylation. The CsA-induced proliferation of human trophoblast cells was effectively abrogated by the EGFR inhibitor AG1478 as well as the ERK inhibitor U0126, but not by the PI3K/PKB inhibitor LY294002. CsA promoted the activation of ERK in JEG-3 cells, which was markedly abrogated in the presence of CXCL12 siRNA, or CXCR4 siRNA, or AG1478. Conclusions CsA may promote EGFR activation via CXCL12/CXCR4 axis, and EGFR downstream ERK signaling pathway may be involved in the CsA-induced proliferation of human trophoblast cells.

E-Cadherin, as a Negative Regulator of Invasive Behavior of Human Trophoblast Cells, Is Down-Regulated by Cyclosporin A Via Epidermal Growth Factor/Extracellular Signal-Regulated Protein Kinase Signaling Pathway

Our previous study has demonstrated cyclosporin A (CsA) promotes the invasiveness of human first-trimester trophoblast cells. In the present study, we further investigated the intracellular signaling pathway responsible for the improvements in CsA-induced invasiveness of human trophoblast cells. We showed that CsA down-regulated E-cadherin transcription and translation in human primary cultured trophoblast cells and choriocarcinoma cell line JEG-3. U0126, an inhibitor of extracellular signal-regulated protein kinase (ERK), attenuated the CsA-induced transcriptional repressor SNAI2 (also called Slug) expression and restored E-cadherin expression inhibited by CsA in JEG-3 cells. We further demonstrated that CsA amplified epidermal growth factor (EGF)-stimulated EGF receptor (EGFR) tyrosine phosphorylation in JEG-3 cells, and inhibition of EGFR tyrosine phosphorylation by AG1478, an EGFR tyrosine kinase inhibitor, abolished the down-regulation of E-cadherin by CsA through ERK signaling pathway. Moreover, our data showed that E-cadherin expression was negatively correlated to the invasiveness of JEG-3 cells, and CsA could reverse the decreased invasiveness of JEG-3 cells that resulted from E-cadherin overexpression. In conclusion, these observations indicate that CsA may decrease E-cadherin expression via EGFR/ERK signaling pathway and, ultimately, contribute to the invasiveness improvement of human trophoblast cells.

Cyclosporin A promotes crosstalk between human cytotrophoblast and decidual stromal cell through up-regulating CXCL12/CXCR4 interaction

BACKGROUND Our previous studies have demonstrated that cyclosporin A (CsA) can increase the cell number in and invasion by human first-trimester trophoblasts and induce maternal-fetal tolerance. C-X-C chemokine receptor type 4 (CXCR4) and C-X-C chemokine ligand 12 (CXCL12) are important mediators at the maternal-fetal interface during early pregnancy. In this study, we further investigate the molecular mechanisms underlying modulation by CsA of the crosstalk between human cytotrophoblast and decidual stromal cell (DSC). METHODS Human first-trimester cytotropoblast and DSC were treated with CsA in the absence or presence of U0126 pretreatment, and then the mRNA and protein levels of CXCL12 and CXCR4 were measured by RT-PCR, qPCR, in-cell western blots and enzyme-linked immunosorbent assay (ELISA), respectively. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and Matrigel invasion assays were used to determine the invasiveness of cytotrophoblast, respectively. The activity of matrix metalloproteinase (MMP)-9 and MMP-2 was detected by gelatin zymography. A co-culture with direct contact between cytotrophoblast and DSC was established and used to investigate the interaction between these two cells. RESULTS CsA up-regulated CXCL12 and CXCR4 expression in human first-trimester cytotrophoblast cells, but not in DSCs. Blocking the mitogen-activated proteinkinase/extracellular signal-regulated kinase (MAPK/ERK1/2) signaling by U0126 abrogated the CsA-induced increase in CXCL12 and CXCR4 expression and neutralizing antibodies to CXCL12 or CXCR4 completely inhibited the CsA-induced increase in cell number, invasion and MMP-9 and MMP-2 activity of cytotrophoblast. CsA also significantly promoted the activity of MMP-9 and MMP-2 in DSCs, but this was unaffected by CXCL12 or CXCR4 neutralizing antibody. Furthermore, the CsA-induced MMP-9 and MMP-2 activity and the invasiveness of cytotrophoblast in the cytotrophoblast and DSC co-culture were significantly increased compared with CsA-treated trophoblast cultured alone, and CXCR4 blocking antibody effectively abolished the increased MMP activity and invasion of cytotrophoblasts in the cytotrophoblast-DSC co-culture stimulated by CsA. CONCLUSIONS CsA can promote the crosstalk between cytotrophoblast and DSC through up-regulating CXCL12/CXCR4 interaction via MAPK signaling, resulting in the increased numbers of and invasion by human cytotrophoblast cells.

Focal Adhesion Kinase Signaling is Necessary for the Cyclosporin A-Enhanced Migration and Invasion of Human Trophoblast Cells

OBJECTIVES Our previous studies have shown that Cyclosporin A (CsA) promotes human trophoblast invasion via mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. E-cadherin and matrix metalloproteinases (MMPs) are important mediators in trophoblast migration and invasion. Here, we further investigate the role of focal adhesion kinase (FAK) signaling in the CsA-induced trophoblast migration and invasion and ERK activation. STUDY DESIGN The migration and invasion of human primary trophoblasts and JEG-3 cells were measured by transwell migration and matrigel invasion assays. The activation of FAK, Src and ERK induced by CsA were examined by western blot. The colocalization of FAK and Src was detected by dual immunofluorescence assay. The regulation of E-cadherin expression and matrix metalloproteinases (MMPs) activity was evaluated by western blot and gelatin zymography, respectively. RESULTS CsA increased the phosphorylation of FAK and Src in human primary trophoblasts and JEG-3 cells. Meanwhile, the activated FAK and Src colocalized in the cytoplasm of JEG-3 cells. The FAK inhibitor Y15 or Src inhibitor PP2 could abrogate the phosphorylation of ERK, the enhanced migration and invasion and the activity of MMP2, 9 induced by CsA. In addition, these inhibitors also restored the expression of E-cadherin which is down-regulated by CsA. However, U0126, an inhibitor of ERK, had no significant effect on the CsA-induced activation of FAK and Src. CONCLUSIONS FAK-Src signaling, the upstream signaling cascade of ERK activation, plays an important role in the CsA-induced migration and invasion via down-regulating expression of E-cadherin and up-regulating activity of MMP2, 9 in trophoblast cells. These results may help provide a rationale to develop a novel therapeutic strategy for pregnancy disorders from insufficient trophoblast invasion.

Chemokine CCL28 induces apoptosis of decidual stromal cells via binding CCR3/CCR10 in human spontaneous abortion.

Spontaneous abortion is the most common complication of pregnancy. Immune activation and the subsequent inflammation-induced tissue injury are often observed at the maternal-fetal interface as the final pathological assault in recurrent spontaneous abortion. However, the precise mechanisms responsible for spontaneous abortion involving inflammation are not fully understood. Chemokine CCL28 and its receptors CCR3 and CCR10 are important regulators in inflammatory process. Here, we examined the expression of CCL28 and its receptors in decidual stromal cells (DSCs) by immunochemistry and flow cytometry (FCM), and compared their expression level in DSCs from normal pregnancy versus spontaneous abortion, and their relationship to inflammatory cytokines production by DSCs. We further analyzed regulation of the pro-inflammatory cytokines on CCL28 expression in DSCs by real-time polymerase chain reaction, In-cell Western and FCM. The effects of CCL28-CCR3/CCR10 interaction on DSC apoptosis was investigated by Annexin V staining and FCM analysis or DAPI staining and nuclear morphology. Higher levels of the inflammatory cytokines interleukin (IL)-1β, IL-17A and tumor necrosis factor-α, and increased CCR3/CCR10 expression were observed in DSCs from spontaneous abortion compared with normal pregnancy. Treatment with inflammatory cytokines differently affected CCL28 and CCR3/CCR10 expression in DSCs. Human recombinant CCL28 promoted DSC apoptosis, which was eliminated by pretreatment with neutralizing antibodies against CCR3/CCR10 and CCL28. However, CCL28 did not affect DSC growth. These results suggest that the inflammation-promoted up-regulation of CCL28 and its receptors interaction in DSCs is involved in human spontaneous abortion via inducing DSC apoptosis.