Yu-Han Meng

CXCL8 enhances proliferation and growth and reduces apoptosis in endometrial stromal cells in an autocrine manner via a CXCR1-triggered PTEN/AKT signal pathway

BACKGROUND Chemokine CXCL8 (also known as IL-8) has been identified as a potential regulator of endometrial stromal cells (ESCs), but it is unclear how CXCL8 regulates the survival of ESCs in the pathogenesis of endometriosis. METHODS We assessed the secretion of CXCL8 by enzyme-linked immunosorbent assays and the expression of its receptors, CXCR1 and CXCR2, by in-cell Western assay and immunohistochemistry. The effects of CXCL8 on the activation or expression of various cell mediators were also investigated by in-cell Western assay. The effects of CXCL8 on the proliferation, growth and apoptosis of ESCs in vitro were assessed by BrdU assays, cell counts and annexin V labeling, respectively. RESULTS Secretion of CXCL8 and expression of CXCR1 in the eutopic ESCs from women with endometriosis were significantly higher than that in control ESCs, but the expression of CXCR2 showed no significant difference between these two cell types. CXCL8 stimulated proliferation and growth and reduced apoptosis of ESCs in an autocrine manner, and these effects were abolished by anti-human CXCL8 and CXCR1 neutralizing antibodies and by a PI3K/Akt inhibitor. Moreover, CXCL8 up-regulated the expression of the anti-apoptotic proteins, survivin and Bcl-2, inhibited the expression of the Phosphatase and tensin homolog (PTEN) and activated the phosphorylation of Akt. CONCLUSIONS This study suggests that CXCL8 and CXCR1 are involved in the pathogenesis of endometriosis by up-regulating proliferation and growth and restricting apoptosis in ESCs by activating the PTEN/Akt pathway and mediating the expression of survivin and Bcl-2.

Chemokine CCL2 enhances survival and invasiveness of endometrial stromal cells in an autocrine manner by activating Akt and MAPK/Erk1/2 signal pathway

OBJECTIVE To clarify the role and mechanism of CCL2 in regulating the biological functions of endometrial stromal cells (ESCs). DESIGN The CCL2 effect on the viability, proliferation, and invasion in the eutopic ESCs from endometriosis. SETTING Research laboratories. PATIENT(S) Patients with endometriosis aged 23-47 years. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Signal transduction and downstream molecules from CCR2. RESULT(S) We have found that the secretion of CCL2 by the eutopic ESCs from endometriosis is higher than that of healthy ESCs without endometriosis. The CCL2 can enhance the viability, proliferation, and invasion of ESCs in a dosage and time-dependent manner. Anti-CCL2 neutralizing antibody and CCR2 antagonist can completely abolish the increase in viability, proliferation, and invasiveness of ESCs induced by CCL2. The CCL2 can increase the expression of proliferating cell nuclear antigen, survivin, and matrix metalloproteinase 2, and decrease the expression of tissue inhibitor of metalloproteinase 1 and 2, and promote the viability, proliferation and invasiveness of ESCs by activating Akt and MAPK/Erk1/2 signal pathway, but not p38 and JNK signal pathway. CONCLUSION(S) CCL2 might play an important role in regulating the functions of ESCs through Akt and MAPK/Erk1/2 signal pathway, and overexpression of CCL2 in ESCs and peritoneal fluid (PF) would lead to onset and development of endometriosis.

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.

CD4 + Foxp3 + regulatory T cell differentiation mediated by endometrial stromal cell-derived TECK promotes the growth and invasion of endometriotic lesions

Endometriosis is associated with an abnormal immune response to endometrial cells, which can facilitate the implantation and proliferation of ectopic endometrial tissue. The proportion of CD4+Foxp3+ regulatory T cells (Tregs) is significantly increased in the peritoneal fluid of women with endometriosis. The thymus-expressed chemokine TECK/CCL25 directly promotes the invasiveness of endometrial stromal cells (ESCs). The aim of this study was to investigate the effects of ESC-derived TECK on the crosstalk between Tregs and ESCs in the progress of endometriosis. We determined that the percentage of Tregs and the concentration of TECK increased in the peritoneal fluid with the progression of endometriosis. The supernatant from co-cultured human ESCs and macrophages not only induced Treg differentiation and increased Treg expression of interleukin-10 (IL-10), transforming growth factor-β (TGF-β) and CD73 by activating the AKT/STAT3 signaling pathway but also repressed Treg apoptosis by downregulating Fas and FasL expression and enhanced the Treg-mediated suppression of CD4+CD25− T cells. In addition, in vitro and in vivo trials confirmed that these effects could be inhibited by anti-TECK neutralizing Abs. The secretion of IL-10 and TGF-β by Tregs increased MMP2 expression and decreased TIMP1 expression and further stimulated the proliferation and invasion of ESCs and the growth of ectopic lesions. These results indicate that TECK derived from ESCs and macrophages upregulates the number and function of Tregs in the ectopic milieu, which contributes to endometriotic immunotolerance and high levels of ESC proliferation and invasion, thereby facilitating the progression of endometriosis.

Cervical Carcinoma Cells Stimulate the Angiogenesis through TSLP Promoting Growth and Activation of Vascular Endothelial Cells

To explore whether cervical carcinomas cells‐derived thymic stromal lymphopoietin (TSLP) modulates the biologic behavior of vascular endothelial cells and herein participates in the angiogenesis in the cervical cancer pathogenesis.

The infiltration and functional regulation of eosinophils induced by TSLP promote the proliferation of cervical cancer cell.

Cervical cancer is often associated with eosinophil (EOS) infiltration, but the source and the role of EOS are still largely unknown. Our previous work has established that thymic stromal lymphopoietin (TSLP) can stimulate the growth of cervical cancer cell in an autocrine manner. Here, we report that EOS infiltration of the lesion site increased gradually with the progression of cervical cancer. The increase in TSLP secretion in HeLa and SiHa cells induced by hypoxia led to a high level of chemokine CCL17 production by HeLa and SiHa cells, and recruited more EOS to the cancer lesion. In addition, TSLP derived from HeLa and SiHa cells promoted proliferation, up-regulated the levels of anti-inflammatory cytokines (IL-10, IL-4, IL-5 and IL-13), and decreased the expression of CD80 and CD86 of EOS. Such educated EOS significantly promoted proliferation and restricted the apoptosis of cervical cancer cells, which was associated with the up-regulation of Ki-67, PCNA and Bcl-2, and the down-regulation of Fas and FasL in HeLa and SiHa cells. These results suggest that a high level of TSLP in cancer lesions mediated by hypoxia is an important regulator of the progression of cervical cancer by recruiting and licensing tumor-associated EOS to promote the growth of the cervical cancer cell itself. This provides a scientific basis on which potential therapeutic strategies could be targeted to cervical cancer, especially for patients with massive infiltrations of EOS.

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.

Estrogen promotes the growth of decidual stromal cells in human early pregnancy

Interleukin-24 (IL-24) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells. The present study aimed to elucidate the biological function of IL-24 and its receptors (IL-20R1, IL-20R2 and IL-22R1) in decidual stromal cells (DSCs) at human maternal-fetal interface. The DSCs behaviors in vitro were verified by viability (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptosis assay, respectively. Additionally, the effects of pregnancy-associated hormones on IL-24 and the effect of IL-24 on the correspondent functional molecules were investigated by ELISA, in-cell western and flow cytometry, respectively. Here we found that DSCs expressed IL-24 and its receptors, and IL-24 obviously suppressed the viability and stimulated the apoptosis in DSCs. On the contrary, both anti-IL-24 and IL-22R1 neutralizing antibodies markedly promoted growth and reduced the apoptosis. Estrogen but not progesterone could significantly decrease IL-24 but not its receptors, and these effects could be abolished by the antagonist of estrogen receptor beta (ERβ). IL-24 significantly restricted the stimulatory effect of estrogen on the viability, anti-apoptosis, anti-apoptosis gene Bcl-2 and proliferation relative gene Ki-67 in DSCs. Our study has demonstrated that IL-24/IL-20R2/IL-22R1 axis is involved in the regulation of estrogen/ERβ signaling on the growth of DSCs through up-regulating the expression of Bcl-2 and Ki67, which suggests that estrogen plays an important role in DSC growth of the early pregnancy through down-regulating IL-24.

RANKL-mediated harmonious dialogue between fetus and mother guarantees smooth gestation by inducing decidual M2 macrophage polarization

Decidual macrophages (dMϕ) contribute to maternal–fetal tolerance. However, the mechanism of dMϕ differentiation during pregnancy is still largely unknown. Here, we report that receptor activator for nuclear factor-κ B ligand (RANKL), secreted by human embryonic trophoblasts and maternal decidual stromal cells (DSCs), polarizes dMϕ toward a M2 phenotype. This polarization is mediated through activation of Akt/signal transducer and activator of transcription 6 (STAT6) signaling, which is associated with the upregulation of histone H3 lysine-27 demethylase Jmjd3 and IRF4 in dMϕ. Such differentiated dMϕ can induce a Th2 bias that promotes maternal–fetal tolerance. Impaired expression of RANKL leads to dysfunction of dMϕ in vivo and increased rates of fetal loss in mice. Transfer of RANK+Mϕ reverses mouse fetal loss induced by Mϕ depletion. Compared with normal pregnancy, there are abnormally low levels of RANKL/RANK in villi and decidua from miscarriage patients. These results suggest that RANKL is a pivotal regulator of maternal–fetal tolerance by licensing dMϕ to ensure a successful pregnancy outcome. This observation provides a scientific basis on which a potential therapeutic strategy can be targeted to prevent pregnancy loss.