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Telocytes enhanced the proliferation, adhesion and motility of endometrial stromal cells as mediated by the ERK pathway in vitro.

ABSTRACT: Telocytes (TCs) is special interstitial cell that have recently been identified in the female reproductive system. Endometriosis (EMs) is a benign gynecological disease whose etiology is still not fully clear. Implantation and proliferation of endometrial stromal cells (ESCs) out of the uterus are essential processes in the development of EMs. Herein, we investigate the in vitro changes of ESCs when cocultured with TCs, and the potential mechanisms involved. The current results demonstrated that, vimentin-positive/pan cytokeratin-negative ESCs, and TCs with a characteristic structure and immunophenotype (CD34/vimentin double-positive) were successfully isolated and harvested. Morphologically, direct cell-to-cell contacts were observed between TCs and ESCs. Quantitatively, TCs treatment clearly promotes the viability of ESCs, enhances cell cycle progression at G2/M phase and upregulates p-ERK1/2 and cyclin-D3 (all P < 0.05). Functionally, ESCs educated by TCs displayed significantly enhanced adhesion ability and accelerated invasion and migration capacity (all P < 0.05). However, no significant changes were found in the rate of apoptosis and in the expression of AKT signaling pathway proteins in TCs-educated ESCs (both P > 0.05). Therefore, TCs treatment obviously enhanced the in vitro motile and invasive capacity of ESCs, which were mediated by the ERK-cyclin-D3 signaling pathway, likely through direct intercellular contacts and/or juxta-paracrine effects; signaling through this axis therefore increased the likelihood of EMs. The enhanced functions of TCs-educated ESCs not only contribute to a deeper understanding of TCs, but also highlight a new concept regarding the physiopathology and therapy of EMs and associated impaired reproductive function.

SUBMITTER: Tang XL

PROVIDER: S-EPMC6413249 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Telocytes enhanced the proliferation, adhesion and motility of endometrial stromal cells as mediated by the ERK pathway in vitro.

Tang Xue-Ling XL Zhang Fei-Lei FL Jiang Xiao-Juan XJ Yang Xiao-Jun XJ

American journal of translational research 20190215 2

Telocytes (TCs) is special interstitial cell that have recently been identified in the female reproductive system. Endometriosis (EMs) is a benign gynecological disease whose etiology is still not fully clear. Implantation and proliferation of endometrial stromal cells (ESCs) out of the uterus are essential processes in the development of EMs. Herein, we investigate the <i>in vitro</i> changes of ESCs when cocultured with TCs, and the potential mechanisms involved. The current results demonstrat ...[more]

PMID: 30899363

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Project description:Extracellular vesicles are highly abundant in seminal fluids and have a known role enhancing sperm function. Clinical pregnancy rates after IVF treatment are improved after female exposure to seminal fluid. Seminal fluid extracellular vesicles (SF-EVs) are candidate enhancers, however, whether SF-EVs interact with cells from the endometrium and modulate the implantation processes is unknown. Here, we investigated whether SF-EVs interact with endometrial stromal cells (ESCs) and enhance decidualisation, a requisite for implantation. SF-EVs, isolated from human seminal fluid (n = 11) by ultracentrifugation, were characterised by nanoparticle tracking analysis and Western blotting, and purified using size exclusion chromatography. Non-decidualised and decidualised primary ESCs (n = 5) were then treated with SF-EVs. Binding of bio-maleimide-labelled SF-EVs was detected by flow cytometry and fluorescence microscopy. Prolactin and IGFBP-1 protein levels in culture media were also analysed after single and multiple SF-EV exposure. SF-EVs size ranged from 50 to 300 nm, and they expressed exosomal markers (ALIX, SYNTENIN-1, CD9 and CD81). SF-EVs bound to non-decidualised and decidualised ESCs at similar levels. ESCs prolactin secretion was increased after single (p = 0.0044) and multiple (p = 0.0021) SF-EV exposure. No differences were found in IGFBP-1 protein levels. In conclusion, SF-EVs enhance in vitro ESC decidualisation and increase secretion of prolactin, an essential hormone in implantation. This elucidates a novel role of SF-EVs on endometrial receptivity. Abbreviations: ECACC: European Collection of Authenticated Cell Cultures; ESCs: endometrial stromal cells; EVs: extracellular vesicles; FCS: foetal calf serum; HRP: horse-radish peroxidase; IFNγ: interferon-gamma; IGF: insulin-like growth factor; IGFBP-1: insulin-like growth factor binding protein 1; IVF: in vitro fertilisation; MVB: multivesicular bodies; NTA: nanoparticle tracking analysis; PRLR-/-: homozygous prolactin receptor knockout; RT: room temperature; SF-EVs: seminal fluid extracellular vesicles; STR: short tandem repeat; TGFβ: transforming growth factor β; uNK: uterine natural killer.

Project description:The enhanced migratory ability of endometrial stromal cells (ESCs) is a key factor in the formation of functional endometrium‑like tissues outside the uterine cavity during endometriosis (EMS). Although accumulating evidence has suggested the importance of microRNAs (miRNAs) in the pathogenesis of EMS, the role of particular miRNAs in the invasiveness of ESCs remain poorly understood. In the present study, the function of miRNAs in the invasiveness of ESCs, along with the associated underlying mechanism involved, were investigated. Initially, the expression patterns of miRNAs in the ectopic and eutopic endometrium isolated from patients with EMS were analyzed using microarray. MicroRNA‑202‑5p (miR‑202) was selected for further study due to its previously reported suppressive effects on the invasion in various types of cancers. The expression of miR‑202 and K‑Ras in eutopic and ectopic endometrioma tissues were detected using reverse transcription‑quantitative PCR, immunohistochemistry and western blotting. The migration and invasion ability of ESCs was determined using wound healing and Transwell invasion assays, respectively. Compared with that from healthy individuals, miR‑202 expression was demonstrated to be lower in the eutopic endometrium from patients with EMS, which was even lower in ectopic endometrium. Functional experiments in primary ESCs revealed that enhanced miR‑202 expression suppressed the cell invasion and migration abilities, which was also accompanied with increased E‑cadherin and reduced N‑cadherin expression in ESCs, suggesting its potentially suppressive role in epithelial‑mesenchymal transition. K‑Ras is a well‑known regulator of the ERK signaling pathway that was shown to be directly targeted and negatively regulated by miR‑202. In addition, K‑Ras expression was found to be upregulated in the ectopic endometrium, where it correlated negatively with that of miR‑202. Knocking down K‑Ras expression mimicked the anti‑invasive effects of miR‑202 overexpression on ESCs, whilst K‑Ras overexpression attenuated the inhibitory role of miR‑202 overexpression in ESC invasion. The K‑Ras/Raf1/MEK/ERK signaling pathway was also blocked by miR‑202 overexpression. These findings suggested that miR‑202 inhibited ESC migration and invasion by inhibiting the K‑Ras/Raf1/MEK/ERK signaling pathway, rendering miR‑202 a candidate for being a therapeutic target for EMS.

Project description:Nucleotide-binding oligomerization domain (NACHT), leucine rich repeat (LRR) and pyrin domain (PYD) 7 containing protein, NLRP7, is a member of the NLR family which serves as innate immune sensors. Mutations and genetic variants of NLRP7 have been found in women with infertility associated conditions, such as recurrent hydatidiform mole, recurrent miscarriage, and preeclampsia. Decidualization of endometrial stromal cells is a hallmark of tissue remodeling to support embryo implantation and proper placental development. Given defective decidualization has been implicated in miscarriage as well as preeclampsia, we aimed to explore the link between the NLRP7 gene and decidualization.Endometrial samples obtained from pregnant women in the first trimester and non-pregnant women were used to study NLRP7 expression pattern. The human telomerase reverse transcriptase (hTERT)-immortalized human endometrial stromal cells (T-HESCs) were used to study the effect of NLRP7 on decidualization. Decidualization of T-HESCs was induced with 1 μM medroxyprogesterone acetate (MPA) and 0.5 mM 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP). siRNA was used to knock down NLRP7 while lentiviral vectors were used to overexpress NLRP7 in cells. NLRP7 expression was detected by immunofluorescence, qRT-PCR, and Western blotting. Decidualization markers, Insulin-like growth factor-binding protein 1 (IGFBP-1) and prolactin (PRL), were detected by qRT-PCR and ELISA. Nuclear translocation of NLRP7 was detected by the subcellular fractionation and confocal microscopy. The effect of NLRP7 on progesterone receptor (PR) activity was evaluated by a reporter system.NLRP7 was up-regulated in the decidual stromal cells of human first-trimester endometrium. After in vitro decidualization, T-HESCs presented with the swollen phenotype and increased expressions of IGFBP-1 and PRL. Knockdown or over-expression of NLRP7 reduced or enhanced the decidualization, respectively, according to the expression level of IGFBP-1. NLRP7 was found to translocate in the nucleus of decidualized T-HESCs and able to promote PR activity.NLRP7 was upregulated and translocated to the nucleus of the endometrial stromal cells in an in vitro decidualization model. Overexpressed NLRP7 promoted the IGFBP-1 expression and PR reporter activation. IGFBP-1 expression decreased with the knockdown of NLRP7. Therefore, we suggest that NLRP7 contributes to in vitro decidualization of endometrial stromal cells.

Dataset Information

Telocytes enhanced the proliferation, adhesion and motility of endometrial stromal cells as mediated by the ERK pathway in vitro.

Publications

Telocytes enhanced the proliferation, adhesion and motility of endometrial stromal cells as mediated by the ERK pathway in vitro.

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