Kisspeptin regulation of human decidual stromal cells motility via FAK–Src intracellular tyrosine kinases

Abstract STUDY QUESTION Can of Clinical Genetics, Maastricht University Medical Centre, Maastricht kisspeptin and its analogues regulate the motility of human decidual stromal cells and what intracellular signaling pathways are involved? SUMMARY ANSWER Kisspeptin analogue–mediated cell motility in human decidual stromal cells via the focal adhesion kinase (FAK)–steroid receptor coactivator (Src) pathway suggesting that kisspeptin may modulate embryo implantation and decidual programming in human pregnancy. WHAT IS KNOWN ALREADY The extravillous trophoblast invades the maternal decidua during embryo implantation and placentation. The motile behavior and invasive potential of decidual stromal cells regulate embryo implantation and programming of human pregnancy. STUDY DESIGN, SIZE, DURATION Human decidual stromal cells were isolated from healthy women undergoing elective termination of a normal pregnancy at 6- to 12-week gestation, after informed consent. PARTICIPANTS/MATERIALS, SETTING, METHODS Kisspeptin analogues were synthetic peptides. Cell motility was estimated by an invasion and migration assay. Immunoblot analysis was performed to investigate the expression of kisspeptin receptor and the effects of kisspeptin analogues on the phosphorylation of FAK and Src. Small interfering RNAs (siRNAs) were used to knock down the expression of kisspeptin receptor, FAK, Src, matrix metallo-proteinases (MMPs) 2 and 9, and extracellular signal-regulated protein kinase (ERK) 1/2. MAIN RESULTS AND THE ROLE OF CHANCE The kisspeptin receptor was expressed in human decidual stromal cells. Kisspeptin agonist decreased, but antagonist increased, cell motility. Kisspeptin agonist decreased the phosphorylation of FAK and Src tyrosine kinases, whereas antagonist increased it. These effects on phosphorylation were abolished by kisspeptin receptor siRNA. The activation of cell motility by kisspeptin analogues was suppressed by siRNA knockdown of endogenous FAK (decreased 66%), Src (decreased 60%), kisspeptin receptor (decreased 26%), MMP-2 (decreased 36%), MMP-9 (decreased 23%), and ERK 1/2 inhibitor (decreased 27%). LIMITATIONS, REASONS FOR CAUTION Human decidual stromal cells were obtained from women having terminations after 6–12 weeks of pregnancy and differences in timing could affect their properties. WIDER IMPLICATIONS OF THE FINDINGS Kisspeptin acting within the endometrium has a potential modulatory role on embryo implantation and decidual programming of human pregnancy. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grant NSC-104-2314-B-182A-146-MY2 (to H.-M.W.) from the Ministry of Science and Technology, Taiwan, and grants CMRPG3E0401 and CMRPG3E0402 (to H.-M.W.). This work was also supported by grants from the Canadian Institutes of Health Research to P.C.K.L. P.C.K.L. is the recipient of a Child & Family Research Institute Distinguished Investigator Award. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER N/A.

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Regulation of Pregnancy: Evidence for Major Roles by the Uterine and Placental Kisspeptin/KISS1R Signaling Systems

Seminars in Reproductive Medicine ◽

10.1055/s-0039-3400966 ◽

2019 ◽

Vol 37 (04) ◽

pp. 182-190 ◽

Cited By ~ 2

Author(s):

Sally Radovick ◽

Andy V. Babwah

Keyword(s):

Stromal Cells ◽

Pregnancy Loss ◽

Therapeutic Agent ◽

Recurrent Pregnancy Loss ◽

Embryo Implantation ◽

Extravillous Trophoblast ◽

Migration And Invasion ◽

Decidual Cell ◽

Signaling Systems ◽

Placental Invasion

AbstractSeveral studies provide strong evidence suggesting that in addition to central kisspeptin/KISS1R signaling, the peripheral uterine- and placental-based kisspeptin/KISS1R signaling systems are major regulators of pregnancy. Specifically, the evidence suggests that the uterine-based system regulates embryo implantation and decidualization, while both the uterine- and placental-based systems regulate placentation. Uterine kisspeptin and KISS1R regulate embryo implantation by controlling the availability of endometrial glandular secretions, like leukemia inhibitory factor, which are essential for embryo adhesion to the uterine epithelium. As for decidualization, the data suggest that decidualized stromal cells express KISS1R and secrete kisspeptin-inhibiting decidual cell motility and thereby indirectly regulate embryo and placental invasion of the uterus. Similarly, for placentation, placental kisspeptin and KISS1R negatively regulate extravillous trophoblast migration and invasion and thereby directly control placental invasion of the uterus. Having recognized a significant role for the uterine- and placental-based kisspeptin/KISS1R signaling systems regulating pregnancy, the future looks promising for the development of kisspeptin and KISS1R as prognostic and diagnostic markers of pregnancy disorders and the use of kisspeptin as a therapeutic agent in the prevention and treatment of conditions such as recurring implantation failure, recurrent pregnancy loss, and preeclampsia.

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Inhibition of TMEM16A impedes embryo implantation and decidualization in mice

Reproduction ◽

10.1530/rep-18-0197 ◽

2018 ◽

Cited By ~ 2

Author(s):

Qianrong Qi ◽

Yifan Yang ◽

Kailin Wu ◽

Qingzhen Xie

Keyword(s):

Progesterone Receptor ◽

Stromal Cells ◽

Embryo Implantation ◽

Mouse Uterus ◽

Endometrial Stromal Cells ◽

Uterine Endometrium ◽

Molecule Inhibitor ◽

Pathway Inhibition ◽

And Function ◽

Reproductive Processes

Recent studies revealed that TMEM16A is involved in several reproductive processes, including ovarian estrogen secretion and ovulation, sperm motility and acrosome reaction, fertilization, and myometrium contraction. However, little is known about the expression and function of TMEM16A in embryo implantation and decidualization. In this study, we focused on the expression and regulation of TMEM16A in mouse uterus during early pregnancy. We found that TMEM16A is up-regulated in uterine endometrium in response to embryo implantation and decidualization. Progesterone treatment could induce TMEM16A expression in endometrial stromal cells through progesterone receptor/c-Myc pathway, which is blocked by progesterone receptor antagonist or the inhibitor of c-Myc signaling pathway. Inhibition of TMEM16A by small molecule inhibitor (T16Ainh-A01) resulted in impaired embryo implantation and decidualization in mice. Treatment with either specific siRNA of Tmem16a or T16Ainh-A01 inhibited the decidualization and proliferation of mouse endometrial stromal cells. In conclusion, our results revealed that TMEM16A is involved in embryo implantation and decidualization in mice, compromised function of TMEM16A may lead to impaired embryo implantation and decidualization.

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The Expression of the Cancer-Associated lncRNA Snhg15 Is Modulated by EphrinA5-Induced Signaling

International Journal of Molecular Sciences ◽

10.3390/ijms22031332 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1332

Author(s):

Daniel Pensold ◽

Julia Gehrmann ◽

Georg Pitschelatow ◽

Asa Walberg ◽

Kai Braunsteffer ◽

...

Keyword(s):

Tyrosine Kinases ◽

Intracellular Signaling ◽

Granule Cells ◽

Cerebellar Granule Cells ◽

Membrane Receptors ◽

In Vitro Assays ◽

Eph Receptor ◽

Medulloblastoma Cell Line ◽

And Migration

The Eph receptor tyrosine kinases and their respective ephrin-ligands are an important family of membrane receptors, being involved in developmental processes such as proliferation, migration, and in the formation of brain cancer such as glioma. Intracellular signaling pathways, which are activated by Eph receptor signaling, are well characterized. In contrast, it is unknown so far whether ephrins modulate the expression of lncRNAs, which would enable the transduction of environmental stimuli into our genome through a great gene regulatory spectrum. Applying a combination of functional in vitro assays, RNA sequencing, and qPCR analysis, we found that the proliferation and migration promoting stimulation of mouse cerebellar granule cells (CB) with ephrinA5 diminishes the expression of the cancer-related lncRNA Snhg15. In a human medulloblastoma cell line (DAOY) ephrinA5 stimulation similarly reduced SNHG15 expression. Computational analysis identified triple-helix-mediated DNA-binding sites of Snhg15 in promoters of genes found up-regulated upon ephrinA5 stimulation and known to be involved in tumorigenic processes. Our findings propose a crucial role of Snhg15 downstream of ephrinA5-induced signaling in regulating gene transcription in the nucleus. These findings could be potentially relevant for the regulation of tumorigenic processes in the context of glioma.

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Low chorionic villous succinate accumulation associates with recurrent spontaneous abortion risk

Nature Communications ◽

10.1038/s41467-021-23827-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xiao-Hui Wang ◽

Sha Xu ◽

Xiang-Yu Zhou ◽

Rui Zhao ◽

Yan Lin ◽

...

Keyword(s):

Spontaneous Abortion ◽

Embryo Implantation ◽

Underlying Mechanism ◽

Recurrent Spontaneous Abortion ◽

Extravillous Trophoblast ◽

Trophoblast Invasion ◽

Iron Sulfur ◽

Succinate Dehydrogenase Complex ◽

Extravillous Trophoblasts ◽

Control Study

AbstractDysregulated extravillous trophoblast invasion and proliferation are known to increase the risk of recurrent spontaneous abortion (RSA); however, the underlying mechanism remains unclear. Herein, in our retrospective observational case-control study we show that villous samples from RSA patients, compared to healthy controls, display reduced succinate dehydrogenase complex iron sulfur subunit (SDHB) DNA methylation, elevated SDHB expression, and reduced succinate levels, indicating that low succinate levels correlate with RSA. Moreover, we find high succinate levels in early pregnant women are correlated with successful embryo implantation. SDHB promoter methylation recruited MBD1 and excluded c-Fos, inactivating SDHB expression and causing intracellular succinate accumulation which mimicked hypoxia in extravillous trophoblasts cell lines JEG3 and HTR8 via the PHD2-VHL-HIF-1α pathway; however, low succinate levels reversed this effect and increased the risk of abortion in mouse model. This study reveals that abnormal metabolite levels inhibit extravillous trophoblast function and highlights an approach for RSA intervention.

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Shp2 in uterine stromal cells critically regulates on time embryo implantation and stromal decidualization by multiple pathways during early pregnancy

PLoS Genetics ◽

10.1371/journal.pgen.1010018 ◽

2022 ◽

Vol 18 (1) ◽

pp. e1010018

Author(s):

Jianghong Cheng ◽

Jia Liang ◽

Yingzhe Li ◽

Xia Gao ◽

Mengjun Ji ◽

...

Keyword(s):

Gene Expression ◽

Signaling Pathways ◽

Stromal Cells ◽

Embryo Implantation ◽

Endometrial Stromal Cells ◽

Conditional Deletion ◽

Enhancer Binding Protein ◽

Epithelial Remodeling ◽

Multiple Signaling ◽

Transcription 3

Approximately 75% of failed pregnancies are considered to be due to embryo implantation failure or defects. Nevertheless, the explicit signaling mechanisms governing this process have not yet been elucidated. Here, we found that conditional deletion of the Shp2 gene in mouse uterine stromal cells deferred embryo implantation and inhibited the decidualization of stromal cells, which led to embryonic developmental delay and to the death of numerous embryos mid-gestation, ultimately reducing female fertility. The absence of Shp2 in stromal cells increased the proliferation of endometrial epithelial cells, thereby disturbing endometrial epithelial remodeling. However, Shp2 deletion impaired the proliferation and polyploidization of stromal cells, which are distinct characteristics of decidualization. In human endometrial stromal cells (hESCs), Shp2 expression gradually increased during the decidualization process. Knockout of Shp2 blocked the decidual differentiation of hESCs, while Shp2 overexpression had the opposite effect. Shp2 knockout inhibited the proliferation of hESCs during decidualization. Whole gene expression profiling analysis of hESCs during the decidualization process showed that Shp2 deficiency disrupted many signaling transduction pathways and gene expression. Analyses of hESCs and mouse uterine tissues confirmed that the signaling pathways extracellular regulated protein kinases (ERK), protein kinase B (AKT), signal transducer and activator of transcription 3 (STAT3) and their downstream transcription factors CCAAT/enhancer binding protein β (C/EBPβ) and Forkhead box transcription factor O1 (FOXO-1) were involved in the Shp2 regulation of decidualization. In summary, these results demonstrate that Shp2 plays a crucial role in stromal decidualization by mediating and coordinating multiple signaling pathways in uterine stromal cells. Our discovery possibly provides a novel key regulator of embryo implantation and novel therapeutic target for pregnancy failure.

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P–322 Addressing progesterone and cAMP signalling pathways for decidualization induction of endometrial stromal cells of patients with endometriosis

Human Reproduction ◽

10.1093/humrep/deab130.321 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

J Moyer ◽

D Dunj. Baston-Buest ◽

G Wennemuth ◽

A Bielfeld ◽

R Grümmer

Keyword(s):

Stromal Cells ◽

Day Treatment ◽

Embryo Implantation ◽

Flow Cytometric Analysis ◽

Prolactin Secretion ◽

In Vivo Model ◽

Endometrial Stromal Cells ◽

Simultaneous Application

Abstract Study question Which compounds/compound combinations are most effective in decidualization induction of endometrial stromal cells (ESCs) of patients with and without endometriosis? Summary answer Combination of compounds addressing different steps in the signalling cascade of decidualization induce decidualization more effectively than application of the individual compounds alone. What is known already Decidualization is the monthly recurring differentiation process of the ESCs in preparation for embryo implantation in human. Undifferentiated ESCs reveal an increased potential to proliferate and invade after retrograde menstruation. This may lead to the formation of ectopic lesions and the manifestation of the chronic gynaecological disease of endometriosis due to an impairment of the decidualization process. Study design, size, duration Compounds and compound combinations addressing the progesterone receptor- or the cAMP-mediated pathway were evaluated with regard to their own and their synergistic potential to induce decidualization of ESCs from women with (n = 10) and without (n = 10) endometriosis during a 6-day treatment. Participants/materials, setting, methods Human primary ESCs were isolated via enzymatic-mechanic digestion from eutopic endometrium from women with and without endometriosis and treated for 6 days in vitro with different progestins (progesterone, medoxyprogesterone acetate (MPA)), 8-Br-cAMP, forskolin, or phosphodiesterase (PDE)-inhibitor (Rolipram) alone or in combination. The degree of decidualization induction was quantified by morphological, biochemical (prolactin) and molecular (HAND2, FOXO1) parameters by means of ELISA, flow cytometric analysis, Realtime PCR and Western blot analysis. Main results and the role of chance After 6 days of treatment, decidualization was induced by forskolin as well as by 8-Br-cAMP whereas progestins or PDE alone hardly induced prolactin secretion by ESCs as a marker of decidualization. A change of morphology from undifferentiated fibroblast-like cells to rounded cells could be observed in parallel with the secretion of prolactin. Forskolin and 8-Br-cAMP-induced decidualization was significantly enhanced by MPA but not by progesterone. These effects were similar in ESCs from women with and without endometriosis. Moreover, forskolin-induced decidualization was significantly enhanced by simultaneous application of PDE. Interestingly, this effect was higher in cells of patients with endometriosis. An induction of decidualization in ESCs was associated with a parallel increase of the process-associated transcription factors HAND2 and FOXO1. This rise of transcription was markedly increased in combination with MPA but not with progesterone. Limitations, reasons for caution Endometrial tissue was obtained from women undergoing infertility treatment and thus may differ from the endometrium of fertile women. Results obtained from primary cells in vitro may not cover the in vivo situation in all respects. Wider implications of the findings: The results of this study provide baseline data for the development of a possible therapeutical approach to induce decidualization as a treatment option for endometriosis. Further research is required to determine the effectiveness of the in vitro tested compound combinations in an in vivo model. Trial registration number Not applicable

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P-322 Addressing progesterone and cAMP signalling pathways for decidualization induction of endometrial stromal cells of patients with endometriosis

Human Reproduction ◽

10.1093/humrep/deab127.077 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

J Moyer ◽

D Dunja Baston-Buest ◽

G Wennemuth ◽

A Bielfeld ◽

R Grümmer

Keyword(s):

Stromal Cells ◽

Day Treatment ◽

Embryo Implantation ◽

Flow Cytometric Analysis ◽

Prolactin Secretion ◽

In Vivo Model ◽

Endometrial Stromal Cells ◽

Simultaneous Application

Abstract Study question Which compounds/compound combinations are most effective in decidualization induction of endometrial stromal cells (ESCs) of patients with and without endometriosis? Summary answer Combination of compounds addressing different steps in the signalling cascade of decidualization induce decidualization more effectively than application of the individual compounds alone. What is known already Decidualization is the monthly recurring differentiation process of the ESCs in preparation for embryo implantation in human. Undifferentiated ESCs reveal an increased potential to proliferate and invade after retrograde menstruation. This may lead to the formation of ectopic lesions and the manifestation of the chronic gynaecological disease of endometriosis due to an impairment of the decidualization process. Study design, size, duration Compounds and compound combinations addressing the progesterone receptor- or the cAMP-mediated pathway were evaluated with regard to their own and their synergistic potential to induce decidualization of ESCs from women with (n = 10) and without (n = 10) endometriosis during a 6-day treatment. Participants/materials, setting, methods Human primary ESCs were isolated via enzymatic-mechanic digestion from eutopic endometrium from women with and without endometriosis and treated for 6 days in vitro with different progestins (progesterone, medoxyprogesterone acetate (MPA)), 8-Br-cAMP, forskolin, or phosphodiesterase (PDE)-inhibitor (Rolipram) alone or in combination. The degree of decidualization induction was quantified by morphological, biochemical (prolactin) and molecular (HAND2, FOXO1) parameters by means of ELISA, flow cytometric analysis, Realtime PCR and Western blot analysis. Main results and the role of chance After 6 days of treatment, decidualization was induced by forskolin as well as by 8-Br-cAMP whereas progestins or PDE alone hardly induced prolactin secretion by ESCs as a marker of decidualization. A change of morphology from undifferentiated fibroblast-like cells to rounded cells could be observed in parallel with the secretion of prolactin. Forskolin and 8-Br-cAMP-induced decidualization was significantly enhanced by MPA but not by progesterone. These effects were similar in ESCs from women with and without endometriosis. Moreover, forskolin-induced decidualization was significantly enhanced by simultaneous application of PDE. Interestingly, this effect was higher in cells of patients with endometriosis. An induction of decidualization in ESCs was associated with a parallel increase of the process-associated transcription factors HAND2 and FOXO1. This rise of transcription was markedly increased in combination with MPA but not with progesterone. Limitations, reasons for caution Endometrial tissue was obtained from women undergoing infertility treatment and thus may differ from the endometrium of fertile women. Results obtained from primary cells in vitro may not cover the in vivo situation in all respects. Wider implications of the findings The results of this study provide baseline data for the development of a possible therapeutical approach to induce decidualization as a treatment option for endometriosis. Further research is required to determine the effectiveness of the in vitro tested compound combinations in an in vivo model. Trial registration number not applicable

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Cell Motility in Microfabricated Models of the Tissue Microenvironment

10.1115/imece2001/bed-23075 ◽

2001 ◽

Author(s):

Kevin Kit Parker ◽

Donald E. Ingber

Keyword(s):

Cell Motility ◽

Signaling Pathways ◽

Intracellular Signaling ◽

Isometric Tension ◽

Migration Behavior ◽

Tissue Microenvironment ◽

Intracellular Signaling Pathways ◽

Capillary Endothelial Cells ◽

Geometric Confinement ◽

Stable Rotation

Abstract We conducted studies using micropatterned substrates to elucidate how cell shape and geometric confinement regulates the inter- and intracellular signaling pathways required for cell motility. When cells were cultured on individual cell-sized square adhesive islands coated with ECM, they extend to the edge of the island and assume a square shape. When these cells were stimulated with growth factors, they preferentially extended lamellipodia from the corners versus the sides. This process was mediated by myosin-generated isometric tension that induced tight spatial localization of Rac in the corners. When two or three capillary endothelial cells are constrained to a fibronectin (FN) island, coordinated cell migration results in stable rotation of the entire system about its center. Thus, the emergent pattern is due to the coordinated migration behavior of the cells. These observations suggest that ECM-induced mechanotransduction potentiates compartmentalized signaling pathways and the multicellular organization required of tissue morphogenesis.

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Seminal plasma enhances and accelerates progesterone-induced decidualisation of human endometrial stromal cells

Reproduction Fertility and Development ◽

10.1071/rd10296 ◽

2012 ◽

Vol 24 (3) ◽

pp. 517 ◽

Cited By ~ 11

Author(s):

U. Doyle ◽

N. Sampson ◽

C. Zenzmaier ◽

P. Schwärzler ◽

P. Berger

Keyword(s):

Seminal Plasma ◽

Stromal Cells ◽

Prolonged Exposure ◽

Embryo Implantation ◽

Combined Treatment ◽

Morphological Differentiation ◽

Endometrial Receptivity ◽

Limiting Factor ◽

Mrna Levels ◽

Endometrial Stromal Cells

In preparation for embryo implantation, endometrial stromal cells (ESC) undergo differentiation, termed decidualisation. Enhancing endometrial decidualisation may overcome reduced endometrial receptivity, a major limiting factor in natural and assisted reproduction. To determine whether seminal plasma (SP) influences decidualisation, primary human ESC were treated with progesterone (P4, 50 ng mL–1) in the presence or absence of dialysed SP (0.5%) for 24 h or for up to 27 days to investigate immediate early effects or the effects of prolonged exposure, respectively. Combined SP and P4 treatment induced ESC morphological differentiation. Relative to control, P4 alone, and SP alone combined treatment with SP and P4 for 27 days significantly upregulated mRNA levels of the decidua-specific markers prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1). Consistently, PRL protein secretion was significantly increased over the course of 27 days combined SP and P4 treatment relative to control, P4 alone and SP alone. Likewise, IGFBP1 secretion was significantly greater relative to control and P4 alone over the course of 27 days. Thus, SP enhances and accelerates P4-mediated decidualisation of human ESC and may enhance endometrial receptivity.

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HB-EGF regulates Prss56 expression during mouse decidualization via EGFR/ERK/EGR2 signaling pathway

Journal of Endocrinology ◽

10.1530/joe-16-0636 ◽

2017 ◽

Vol 234 (3) ◽

pp. 247-254 ◽

Cited By ~ 7

Author(s):

Jie Liu ◽

Fei Gao ◽

Yue-Fang Liu ◽

Hai-Ting Dou ◽

Jia-Qi Yan ◽

...

Keyword(s):

Signaling Pathway ◽

Stromal Cells ◽

Embryo Implantation ◽

Implantation Site ◽

Mouse Uterus ◽

Delayed Implantation ◽

Initial Stage ◽

And Function ◽

Key Steps

Embryo implantation and decidualization are key steps for successful reproduction. Although numerous factors have been identified to be involved in embryo implantation and decidualization, the mechanisms underlying these processes are still unclear. Based on our preliminary data, Prss56, a trypsin-like serine protease, is strongly expressed at implantation site in mouse uterus. However, the expression, regulation and function of Prss56 during early pregnancy are still unknown. In mouse uterus, Prss56 is strongly expressed in the subluminal stromal cells at implantation site on day 5 of pregnancy compared to inter-implantation site. Under delayed implantation, Prss56 expression is undetected. After delayed implantation is activated by estrogen, Prss56 is obviously induced at implantation site. Under artificial decidualization, Prss56 signal is seen at the primary decidual zone at the initial stage of artificial decidualization. When stromal cells are induced for in vitro decidualization, Prss56 expression is significantly elevated. Dtprp expression under in vitro decidualization is suppressed by Prss56 siRNA. In cultured stromal cells, HB-EGF markedly stimulates Prss56 expression through EGFR/ERK pathway. Based on promoter analysis, we also showed that Egr2 is involved in Prss56 regulation by HB-EGF. Collectively, Prss56 expression at implantation site is modulated by HB-EGF/EGFR/ERK signaling pathway and involved in mouse decidualization.

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