IL-1β up-regulates expression of IL-8 in endometrial stromal cells in vitro

Endometriosis(EMs),which presents a majorchallenge to gynecologist,is a common disease amongreproductive-aged women and its morbidity increases inrecent years.Besides genetic,hormonal,and environ-mental factors involved in the development,many stud-ies on endometriosis indicate that there are more cellsrelevant to the immunoinflammatory process and vari-ous cytokines infiltrating into ectopic and eutopic endo-metrium of endometriosis than into endometrium ofhealthy women[1].IL-1β,one of the …     

The presence of heparins during decidualization modulates the response of human endometrial stromal cells to IL-1β in vitro

Purpose

The aim of this paper is to study the impact of heparin on the response of human endometrial stromal cells (ESCs) to interleukin (IL)-1β during decidualization in vitro.

Methods

ESCs were isolated from hysterectomy specimens of premenopausal women undergoing hysterectomy for benign reasons; decidualized in vitro and incubated in parallel with unfractionated heparin or tinzaparin; and stimulated with IL-1β at days 0, 3, 6, and 9 during decidualization. IL-6, IL-11, and leukemia inhibitory factor (LIF) were analyzed using ELISAs and real-time RT-PCR. Cell viability was determined by a fluorometric assay.

Results

IL-1β dose-dependently stimulated IL-6, IL-11, and LIF in distinct patterns in ESCs during decidualization. Unfractionated heparin as well as tinzaparin attenuated the IL-1β-mediated induction of IL-6, IL-11, and LIF on protein and messenger RNA (mRNA) levels. The relative effects of heparin and tinzaparin were getting more pronounced during the time course of decidualization.

Conclusions

Unfractionated heparin and the low molecular weight heparin tinzaparin have modulating effects on IL-1β-induced endometrial cytokines of the IL-6 family during decidualization. These effects of heparins beyond their classical anti-coagulatory properties might have implications on the regulation of endometrial receptivity and early implantation.

     

Normal endometrial stromal cells regulate 17β-estradiol-induced epithelial-mesenchymal transition via slug and E-cadherin in endometrial adenocarcinoma cells in vitro

Stroma–tumor communication participates in the pathogenesis of endometrial carcinomas. In previous studies, we found that normal stromal cells inhibited the growth of endometrial carcinoma cells. Here, we investigated the role of normal stromal cells in the epithelial-mesenchymal transition (EMT) of endometrial carcinoma cells and explored the possible mechanism implied. We found that conditioned medium (CM) by normal endometrial stromal cells (NSC) reduced cell growth and induced cell apoptosis in Ishikawa cells. CM by NSC inhibited 17β-estradiol-induced cell growth and apoptosis decrease in Ishikawa cells. Moreover, CM by NSC inhibited the migration and invasion, and 17β-estradiol-induced migration and invasion in Ishikawa cells. Meanwhile, CM by NSC decreased Slug expression and 17β-estradiol-induced Slug expression, increased E-cadherin expression and abolished 17β-estradiol-induced E-cadherin reduction in Ishikawa cells. In conclusion, normal stromal factors can inhibit 17β-estradiol-induced cell proliferation and apoptosis inhibition, and abolished 17β-estradiol-induced EMT in endometrial cancer cell via regulating E-cadherin and Slug expression.     

Does metformin influence the insulin-, IGF I- and IGF II-receptor gene expression and Akt phosphorylation in human decidualized endometrial stromal cells?

Objective

To assess the effects of metformin on insulin-, IGF I-, and IGF II-receptor gene expression and Akt phosphorylation in decidualized human endometrial stromal cells (ESC) after stimulation with insulin, IGF I and II.

Study design

ESC were isolated from healthy, regularly cycling women and after two passages decidualized with estrogen/progesterone ± metformin. Cells were incubated with insulin, IGF I or IGF II for 1, 5, and 10 min to assess Akt phosphorylation by Western blot. To investigate the insulin-, IGF I- and IGF II-receptor gene expression ESC were incubated with insulin, IGF I or IGF II for 6 and 24 h.

Results

Insulin- and IGF I-receptor gene expression in ESC changed significantly after incubation with insulin, IGF I or IGF II. This was further augmented in metformin pretreated cells, while IGF II-receptor gene expression changed particularly after pretreatment with metformin. Akt phosphorylation peaked after 5 min insulin, IGF I and IGF II stimulation in ESC in both control (control 0.08 ± 0.03 vs. insulin 0.74 ± 0.19, IGF I 0.68 ± 0.22, IGF II 0.53 ± 0.13, p < 0.05) and metformin pretreated cells (control 0.03 ± 0.01 vs. insulin 0.75 ± 0.11, IGF I 0.74 ± 0.15, IGF II 0.67 ± 0.09, p < 0.005). However, there was no significant difference between the control and metformin pretreated group.

Conclusion

Insulin, IGF I and IGF II lead to changes in their receptor gene expression and induced Akt phosphorylation in ESC. These effects were further highlighted in the presence of metformin.     

Endometriotic epithelial cells induce MMPs expression in endometrial stromal cells via an NFкB-dependent pathway

Objective.?To explore the stroma–epithelium interactions in endometriosis and to identify the possible signalling pathways involved in this cross-talk.

Design.?Laboratory study via primary cultured endometrial stromal and epithelial cells.

Setting.?University Hospital.

Patients.?Fifteen patients with endometriosis confirmed by histopathology were recruited in the study, and 12 women free of endometriosis were used as control group.

Intervention(s).?Specific NFкB inhibitor 1-Pyrrolidinecarbodithioic acid ammonium salt (PDTC) was used in cell cultures.

Main outcome measure(s).?The expression and secretion of MMP-2, MMP-9, TIMP-1, TIMP-2 and the DNA-binding activity of NFкB in normal endometrial stromal cells or in co-cultures with normal or endometriotic epithelial cells from patients with endometriosis.

Result(s).?Endometrial epithelial cells induced MMP-9 and MMP-2 expression in normal stromal cells in vitro. In co-cultures with endometriotic epithelial cells, normal endometrial stromal cells expressed and secreted higher MMP-2 (p?<?0.05) and MMP-9 (p?<?0.05). Specific inhibition of NFкB pathway in stromal cells abolished this induction effect by epithelial cells.

Conclusion(s).?Endometriotic epithelial cells induce MMPs expression and secretion in normal endometrial stromal cells via an NFкB-dependent pathway in vitro. This cross-talk between epithelial cells and stromal cells may facilitate the implantation and extension of the ectopic foci and favour the development of the disease.     

In vitro effects of peroxisome proliferator-activated receptor-γ ligands on gene expression in lipopolysaccharide-induced endometrial and endometriotic stromal cells

This in vitro study demonstrates the comparative anti-inflammatory effects of rosiglitazone and 15d-PGJ(2) in endometriosis and provides evidence for exploitation of peroxisome proliferator-activated receptor-γ as a therapeutic target.     

Are growth factor receptors modulated by metformin in human endometrial stromal cells after stimulation with androgen and insulin?

Objective

To assess the effect of metformin on gene and protein expression of insulin receptor (IR) and IGF-1 (IGF-1R) receptor in human endometrial stromal cells after stimulation with androgen and insulin.

Study design

Primary culture of endometrial stromal cells stimulated with estrogen, progesterone with or without androgen or insulin, and treated with metformin for 24 and 48 h, followed by RNA (qRT-PCR) and protein (Western blot) extraction and analysis.

Results

IR gene expression was increased after treatment with insulin (2.9-fold change, p = 0.027) and further after metformin treatment (4.7-fold change, p < 0.001), and in IGF-1R, the group treated with insulin (1.83-fold change) and metformin (1.78-fold change) showed more expression, than control group (p < 0.001). Similarly, IR protein expression was increased after addition of metformin and insulin (249,869 ± 15,878) in relation to the other groups (p < 0.001). Furthermore, cells treated with insulin (153,634 ± 29,123) and androgen plus insulin (162,854 ± 86,258) had a higher IR protein expression compared to control (104,654 ± 5,634) and androgen group (71,595 ± 3,439, (p = 0.045 and 0.021). In groups treated with insulin (127,711 ± 4,591) and androgen plus insulin (151,098 ± 5,194) the protein IGF-1R was increased compared to control (79,355 ± 3,470) and the androgen-only group (79,326 ± 3,114) (p < 0.001).

Conclusion

Metformin in combination with insulin increased IR protein and gene expressions, while it had no influence on the protein expression of IGF-1R in endometrial stromal cells.     

Dose–response effect of interleukin (IL)-1β, tumour necrosis factor (TNF)-α, and interferon-γ on the in vitro production of epithelial neutrophil activating peptide-78 (ENA-78), IL-8, and IL-6 by human endometrial stromal cells

Purpose  

The production of epithelial neutrophil activating peptide-78 (NA-78) and the interleukins IL-8 and IL-6 by endometrial stromal cells is stimulated by pro-inflammatory interleukin-1 (IL-1) and tumour necrosis factor-α (TNF-α). IL-8 is suggested to play a role in the pathogenesis of endometriosis, and in these women the peritoneal fluid concentrations of ENA-78 and IL-8 are increased. TNF-α has been tested together with interferon-γ because of their cooperative stimulation of IL-6. The release of IL-8, however, is inhibited with increasing interferon levels. The aim of the study was the analysis of the production of ENA-78, IL-6 and IL-8 by cultured human endometrial stromal cells in the presence of varying concentrations of IL-1β, TNF-α, and interferon-γ.     

ILK enhances migration and invasion abilities of human endometrial stromal cells by facilitating the epithelial–mesenchymal transition

Abstract

Epithelial–mesenchymal transition (EMT) plays a significant part in the pathogenesis of endometriosis by facilitating the migration and invasion abilities of cells. Integrin-linked kinase (ILK) increases the cell migration and invasion abilities by inducing the EMT. Eutopic and control endometrial stromal cells (EuSCs and CSCs) were isolated and cultured. Cell migration and invasion abilities were detected by transwell assays. Levels of proteins were detected by Western blot. EuSCs showed higher levels of ILK, N-cadherin, vimentin and stronger migration and invasion abilities. After transfection of siRNA-ILK, E-cadherin and keratin levels were increased while N-cadherin and vimentin levels were decreased in EuSCs. Besides that, the migration and invasion abilities of EuSCs were significantly decreased after transfection of siRNA-ILK. On the contrary, levels of ILK, N-cadherin and vimentin were increased while levels of E-cadherin and keratin were decreased simultaneously after transfecting CSCs with pEGFP-C1-ILK. Simultaneously, the migration and invasion abilities of CSCs were increased after transfection of pEGFP-C1-ILK. Our study verified that high expression of ILK enhanced the migration and invasion abilities of ESCs by facilitating the EMT. Given that ILK played crucial roles in the pathogenesis of endometriosis, it may be considered as a promising targeted therapy for endometriosis.     

Interleukin-13 and tumor necrosis factor-β differentially regulate the production of cytokines by cultured human endometrial stromal cells

ObjectiveTo evaluate the effects of interleukin (IL)-13, a T-helper (Th)2 cytokine, and tumor necrosis factor (TNF)-β, a Th1 cytokine, on the production of IL-6 family cytokines and chemokines by endometrial stromal cells (ESC).DesignThe effects of IL-13 and TNF-β, on the production of IL-6, IL-11, leukemia inhibitory factor (LIF), IL-8, growth-regulated oncogene α (GROα), monocyte chemoattractant protein-1 (MCP-1), regulated on activation, T-cell expressed and secreted (RANTES), and eotaxin were investigated.SettingResearch laboratory at a medical university.Patient(s)Thirteen endometrial specimens in the late proliferative phase were used.Intervention(s)The ESC were incubated for 24 hours with recombinant human IL-13 and recombinant human TNF-β.Main outcome measure(s)The concentration of IL-6, IL-11, LIF, IL-8, GROα, MCP-1, RANTES, and eotaxin in the culture media was measured using ELISA.Result(s)The increase in levels of IL-6, IL-8, MCP-1, and eotaxin in the culture media of ESC paralleled the addition of increasing amounts of IL-13 and TNF-β, whereas the levels of IL-11 and LIF were decreased with increasing amounts of IL-13, but were increased with increasing amounts of TNF-β. Tumor necrosis factor-β enhanced the production of GROα and RANTES in dose-dependent manner; however, IL-13 did not affect the expression of GROα or RANTES.Conclusion(s)These results suggest that IL-13 and TNF-β secreted in the cyclic endometrial tissue and in the decidua may differentially regulate the production of IL-6 family cytokines and chemokines by ESC. The controlled expression of these cytokines in the endometrium may contribute to the modulation of the immune reaction during the menstrual cycle and in early pregnancy by the regulation of leukocyte trafficking and functions.     

Dexamethasone stimulates the expression of leptin and 11β-HSD2 in primary human placental trophoblastic cells

Objectives

Fetal glucocorticoid excess is thought to play an important role in early-life programming, promoting growth restriction and contributing to adult metabolic, cardiovascular and neuroendocrine disease. We hypothesized that dexamethasone incubation of primary trophoblastic cells from human healthy placentas at term might induce altered gene and protein expression of several endocrine placental regulators.

Study design

Primary villous trophoblastic cells were incubated with 10 μM dexamethasone for 6, 12, 24, 48 and 72 h. Non-incubated trophoblastic cells served as vehicle control. Gene expression of leptin, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) and insulin-like growth factor-binding protein-1 (IGFBP-1) was measured. Moreover, leptin, β-human chorionic gonadotropine (β-hCG) and lactate dehydrogenase (LDH) release into the culture medium was determined.

Results

Leptin gene expression was significantly increased in dexamethasone-incubated trophoblastic cells after 24, 48 and 72 h. There was a significant increase in leptin concentration in the medium of the cell culture after 48 h. Gene expression of 11β-HSD2 was significantly higher in dexamethasone-stimulated trophoblastic cells compared to vehicle controls after 72 h. The expression rate of IGFBP-1 mRNA was basal throughout the incubation period. The concentration of β-HCG in the supernatant increased significantly after 72 h of dexamethasone incubation, while LDH concentrations remained stable.

Conclusion

Our findings suggest that dexamethasone incubation stimulates leptin and 11β-HSD2 gene expression in primary villous trophoblastic cells of healthy human placentas, while enhancing cytotrophoblast differentiation.     

Knockdown of E-cadherin expression of endometrial epithelial cells may activate Wnt/β-catenin pathway in vitro

Purpose

E-cadherin, a transmembrane glycoprotein mediating Ca²⁺-independent homotypic cell–cell adhesion in epithelial cell, plays an essential role in metastasis. It has been postulated that E-cadherin downregulation is a crucial mechanism in the pathogenesis of endometriosis. To evaluate the effect on the cell behavior after knockdown of E-cadherin gene (CDH1) in cultured human endometrial epithelial cells (EECs) isolated from normal endometrium.

Methods

EECs were isolated from the endometrial tissues of fertile woman who underwent total hysterectomy due to cervical intraepithelial neoplasia III. CDH1 expression was knocked down by small hairpin RNA. The EECs transfected with empty vector served as control. Transwell assay was used to test EECs migration or invasion. qRT-PCR and western blot were used to detect mRNA and protein levels.

Results

The results showed that knockdown of E-cadherin expression can increase cell migration and invasion, and up-regulate mRNA and protein levels of β-catenin, cyclinD1, and c-myc.

Conclusions

Down-regulation of E-cadherin expression may activate the Wnt/β-catenin pathway in endometrial cells, which may together participate in the occurrence of endometriosis.

     

Expression of adrenomedullin in human ovaries,ovarian sex cord–stromal tumors and cultured granulosa–luteal cells

The aim of the present study was to characterise the expression pattern of the multifunctional vasoactive peptide adrenomedullin (ADM) in human ovarian tumors, and to find hormonal regulators of ADM expression in human ovaries. The expression of ADM messenger RNA (mRNA) was higher in granulosa cell tumors than in fibrothecomas and normal ovaries, as analysed by Northern blots. In normal ovaries, ADM immunoreactivity was localised in both granulosa and thecal cells. Eight of the 90 granulosa cell tumors (9%) showed moderate and 53 (59%) weak ADM immunoreactivity, whereas 27% (11/41) of the fibrothecomas displayed weak ADM staining. FSH, protein kinase A activator (Bu)₂cAMP, prostaglandin E₂ (PGE₂), activin A and the broad protein kinase regulator staurosporine decreased ADM mRNA accumulation in cultured granulosa–luteal cells time- and dose-dependently. FSH, (Bu)₂cAMP and PGE₂ increased progesterone secretion and the accumulation of the steroidogenic acute regulatory protein mRNA in these cells. In conclusion, ADM is expressed in normal human ovaries and sex cord–stromal tumors, particularly in those of granulosa cell origin. FSH, PGE_2, (Bu)₂cAMP and activin A suppress ADM gene expression in granulosa–luteal cells. Expression of ADM in human ovaries and its hormonal regulation in granulosa cells suggests a paracrine role for ADM in ovarian function.