Removal of Rev-erbα inhibition contributes to the prostaglandin G/H synthase 2 expression in rat endometrial stromal cells

2015 ◽  
Vol 308 (8) ◽  
pp. E650-E661 ◽  
Author(s):  
Keishiro Isayama ◽  
Lijia Zhao ◽  
Huatao Chen ◽  
Nobuhiko Yamauchi ◽  
Yasufumi Shigeyoshi ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Chromatin Immunoprecipitation ◽  
Clock Genes ◽  
Pcr Analysis ◽  
Core Component ◽  
Endometrial Stromal Cells ◽  
Transcript Levels ◽  
Rhythmic Expression ◽  
Decidual Cells ◽  
Implantation Sites

The rhythmic expression of clock genes in the uterus is attenuated during decidualization. This study focused on Ptgs2, which is essential for decidualization, as a putative clock-controlled gene, and aimed to reveal the functions of clock genes in relation to Ptgs2 during decidualization. We compared the transcript levels of clock genes in the rat uterus on days 4.5 (D4.5) and 6.5 of pregnancy. The transcript levels of clock genes ( Per2, Bmal1, Rorα, and Rev-erbα) had decreased at implantation sites on day 6.5 (D6.5e) compared with those on D4.5, whereas Ptgs2 transcripts had increased on D6.5e. Similar observations of Rev-erbα and Ptgs2 were also obtained in the endometrium on D6.5e by immunohistochemistry. In the decidual cells induced by medroxyprogesterone and 2- O-dibutyryl-cAMP, the rhythmic expression levels of clock genes were attenuated, whereas Ptgs2 transcription was induced. These results indicate that decidualization causes the attenuation of clock genes and the induction of Ptgs2. Furthermore, in the experiment of Bmal1 siRNA, the rhythmic expression of clock genes and Ptgs2 was attenuated by the siRNA. Transcript levels of Ptgs2 and prostaglandin (PG)E2 production were increased by treatment with the Rev-erbα antagonist, suggesting the contribution of the nuclear receptor Rev-erbα to Ptgs2 expression. Moreover, Rev-erbα knockdown enhanced the induction of Ptgs2 transcription and PGE2 production by forskolin. Chromatin immunoprecipitation-PCR analysis revealed that Rev-erbα could directly bind to a proximal RORE site of Ptgs2. Collectively, this study demonstrates that the attenuation of the circadian clock, especially its core component Rev-erbα, contributes to the induction of Ptgs2 during decidualization.

Inhibitory role of REV-ERBα in the expression of bone morphogenetic protein gene family in rat uterus endometrium stromal cells

2015 ◽  
Vol 308 (7) ◽  
pp. C528-C538 ◽  
Author(s):  
Hirotaka Tasaki ◽  
Lijia Zhao ◽  
Keishiro Isayama ◽  
Huatao Chen ◽  
Nobuhiko Yamauchi ◽  
...  
Keyword(s):  
Gene Family ◽  
Bone Morphogenetic Proteins ◽  
Stromal Cells ◽  
Clock Genes ◽  
Pcr Analysis ◽  
Gene Promoters ◽  
Endometrial Stromal Cells ◽  
Morphogenetic Protein ◽  
Decidual Cells

Uterus circadian rhythms have been implicated in the gestation processes of mammals through entraining of the clock proteins to numerous downstream genes. Bone morphogenetic proteins (BMPs), having clock-controlled regulatory sites in their gene promoters, are expressed in the uterus during decidualization, but the regulation of the Bmp gene expression is poorly understood. The present study was designed to dissect the physiological roles of the uterus oscillators in the Bmp expression using the uterus endometrial stromal cells (UESCs) isolated from Per2-dLuc transgenic rats on day 4.5 of gestation. The in vitro decidualization of UESCs was induced by medroxyprogesterone acetate and 2-O-dibutyryl cAMP. A significant decline of Per2-dLuc bioluminescence activity was induced in decidual cells, and concomitantly, the expression of canonical clock genes was downregulated. Conversely, the expression of the core Bmp genes Bmp2, Bmp4, Bmp6, and Bmp7 was upregulated. In UESCs transfected with Bmal1-specific siRNA, in which Rev-erbα expression was downregulated, Bmp genes, such as Bmp2, Bmp4, and Bmp6 were upregulated. However, Bmp1, Bmp7, and Bmp8a were not significantly affected by Bmal1 silencing. The expression of all Bmp genes was enhanced after treatment with the REV-ERBα antagonist (SR8278), although their rhythmic profiles were differed from each other. The binding of REV-ERBα to the proximal regions of the Bmp2 and Bmp4 promoters was revealed by chromatin immunoprecipitation-PCR analysis. Collectively, these results indicate that the Bmp genes are upregulated by the attenuation of the cellular circadian clock; in particular, its core component REV-ERBα functions as a transcriptional silencer in the Bmp gene family.

scholarly journals Progesterone-Dependent Induction of Phospholipase C-Related Catalytically Inactive Protein 1 (PRIP-1) in Decidualizing Human Endometrial Stromal Cells

Endocrinology ◽  
2016 ◽  
Vol 157 (7) ◽  
pp. 2883-2893 ◽  
Author(s):  
Joanne Muter ◽  
Paul J. Brighton ◽  
Emma S. Lucas ◽  
Lauren Lacey ◽  
Anatoly Shmygol ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Stromal Cells ◽  
Nuclear Translocation ◽  
Scaffold Protein ◽  
Trophoblast Invasion ◽  
Differentiation Markers ◽  
Endometrial Stromal Cells ◽  
Decidual Cells ◽  
Phosphoinositide 3 Kinase ◽  
Inactive Protein

Decidualization denotes the transformation of endometrial stromal cells into specialized decidual cells. In pregnancy, decidual cells form a protective matrix around the implanting embryo, enabling coordinated trophoblast invasion and formation of a functional placenta. Continuous progesterone (P4) signaling renders decidual cells resistant to various environmental stressors, whereas withdrawal inevitably triggers tissue breakdown and menstruation or miscarriage. Here, we show that PLCL1, coding phospholipase C (PLC)-related catalytically inactive protein 1 (PRIP-1), is highly induced in response to P4 signaling in decidualizing human endometrial stromal cells (HESCs). Knockdown experiments in undifferentiated HESCs revealed that PRIP-1 maintains basal phosphoinositide 3-kinase/Protein kinase B activity, which in turn prevents illicit nuclear translocation of the transcription factor forkhead box protein O1 and induction of the apoptotic activator BIM. By contrast, loss of this scaffold protein did not compromise survival of decidual cells. PRIP-1 knockdown did also not interfere with the responsiveness of HESCs to deciduogenic cues, although the overall expression of differentiation markers, such as PRL, IGFBP1, and WNT4, was blunted. Finally, we show that PRIP-1 in decidual cells uncouples PLC activation from intracellular Ca2+ release by attenuating inositol 1,4,5-trisphosphate signaling. In summary, PRIP-1 is a multifaceted P4-inducible scaffold protein that gates the activity of major signal transduction pathways in the endometrium. It prevents apoptosis of proliferating stromal cells and contributes to the relative autonomy of decidual cells by silencing PLC signaling downstream of Gq protein-coupled receptors.

266. Supressor of cytokine signalling 3 regulates IL-11 induced human endometrial stromal cell decidualization

2005 ◽  
Vol 17 (9) ◽  
pp. 109
Author(s):  
E. Dimitriadis ◽  
C. Stoikos ◽  
L. A. Salamonsen
Keyword(s):  
Stromal Cells ◽  
Embryo Implantation ◽  
Cellular Protein ◽  
Pcr Analysis ◽  
Cytokine Signaling ◽  
Pituitary Cells ◽  
Human Pituitary ◽  
Endometrial Stromal Cells ◽  
Socs3 Protein

Decidualization of endometrial stromal cells is critical for embryo implantation and establishment of pregnancy. Locally produced cytokines such as interleukin (IL)-11 enhance decidualization of human endometrial stromal cells (HESC). IL-11 signaling is negatively regulated by suppressor of cytokine signaling (SOCS) proteins. IL-11 stimulates SOCS3 in human pituitary cells. The aim of this study was to examine the role of SOCS3 on IL-11 induced HESC decidualization. Decidualization of HESC was assessed using an in vitro model in which estrogen (E)+progesterone (P) or cAMP was administered for 8 days to cells. Medium was collected for prolactin (PRL) assay (a decidual marker). Cellular protein was extracted for Western analysis and cellular RNA for real-time RT-PCR analysis. SOCS3 was overexpressed in HESC cells and the effect on decidualization assessed. HESC treated with E+P or cAMP secreted PRL from day 6. Treatment of HESC with E+P or cAMP increased the abundance of SOCS3 protein, coinciding with an increase in PRL secretion. cAMP maximally stimulated SOCS3 protein and mRNA during decidualization. Antiprogestin (onapristone) added to E+P or cAMP treated cells at day 6 reduced PRL secretion but had no influence on SOCS3 abundance suggesting that SOCS3 protein was not regulated via the P-receptor pathway. Addition of IL-11 to HESC increased SOCS3 abundance from 1 h. SOCS3 abundance returned to control levels following treatment of cells with IL-11 and IL-11 neutralising antibody. SOCS3 overexpression in HESC treated with cAMP reduced PRL secretion compared to mock- or non-transfected HESC. Furthermore, IL-11 mediated decidualization was diminished by SOCS3 overexpression. We have demonstrated for the first time that SOCS3 regulates IL-11 induced decidualization and that SOCS3 overexpression in HESC disrupts decidualization. This knowledge is important in understanding the mechanisms by which IL-11 promotes decidualization of HESC and thus the formation of decidua, an essential component of a functional placenta.

414. The contraceptive potential of a long-acting IL-11 inhibitor

2008 ◽  
Vol 20 (9) ◽  
pp. 94
Author(s):  
E. Menkhorst ◽  
L. Salamonsen ◽  
L. Robb ◽  
E. Dimitriadis
Keyword(s):  
Nature Medicine ◽  
Embryo Implantation ◽  
Hormonal Contraceptive ◽  
Cyclin D3 ◽  
Human Reproduction ◽  
Luminal Epithelium ◽  
Endometrial Stromal Cells ◽  
Decidual Cells ◽  
Implantation Sites ◽  
Long Acting

Interleukin 11 (IL-11) signalling is essential for the establishment of pregnancy in mice, through its action on the differentiation of uterine endometrial stromal cells (decidualisation), a critical process during embryo implantation. IL-11Rα deficient mice are infertile due to defective decidualisation1. IL-11 expression peaks between days (D) 4.5–9.5 of pregnancy (D0: day of plug) in mouse decidua. We examined the effect of administering (intraperitoneal [IP] injection or vaginal gel) a PEGylated IL-11 antagonist (PEGIL-11A) on decidualisation and pregnancy outcome in mice. The sera half-life of PEGIL-11A (IC50 2.8nM) following IP injection was 24h, compared with <1 h for the non-PEGylated antagonist (IC50 0.26nM). Following IP injection, PEGIL-11A localised to decidual cells and blocked the IL-11 decidual target protein, cyclin D3. IP injection of 600µg/application PEGIL-11A (or PEG control) at 1000 h and 1600 h on D3 and 1000 h on D4 (n = 4/group), resulted in smaller implantation sites than controls on D6 due to retarded mesometrial decidual formation. On D10, severe decidual destruction was visible: implantation sites contained regions of haemorrhage and the uterine luminal epithelium had reformed, suggesting a return to oestrous cycling. Following vaginal application in aqueous placebo gel, PEGIL-11A localised to decidual cells. Vaginal application of 200µg/application PEGIL-11A (or control) twice daily from D2 to D5 (n = 4/group), resulted in smaller implantation sites than controls on D6 due to partial inhibition of mesometrial decidual formation. This study demonstrates that PEGIL-11A blocked IL-11 action in the uterus, resulting in total pregnancy loss, equivalent to the IL-11Rα deficient mouse. In women, IL-11 and its receptor are produced by the uterine luminal and glandular epithelium during the period of uterine receptivity2, suggesting that IL-11 may act during initial blastocyst attachment to the luminal epithelium as well as stromal decidualisation. This study provides proof-of-principle for the development of a novel, non-hormonal contraceptive for women. (1) Robb L et al. Nature Medicine 1998; 4: 303–308. (2) Dimitriadis E et al. Molecular Human Reproduction 2000; 6: 907–914.

scholarly journals Expression of Wilms’ Tumor Suppressor Gene (WT1) in Human Endometrium: Regulation through Decidual Differentiation

2001 ◽  
Vol 86 (12) ◽  
pp. 5964-5972
Author(s):  
Antonis Makrigiannakis ◽  
George Coukos ◽  
Anastasia Mantani ◽  
Prokopis Prokopakis ◽  
Geoffrey Trew ◽  
...  
Keyword(s):  
Menstrual Cycle ◽  
Stromal Cells ◽  
Wilms Tumor ◽  
Suppressor Gene ◽  
Secretory Phase ◽  
Endometrial Stromal Cells ◽  
Protein Levels ◽  
Decidual Cells ◽  
Wt1 Protein

The Wilms’ tumor suppressor gene (WT1) encodes a zinc-finger containing transcription factor that is selectively expressed in the developing urogenital tract and functions as a tissue-specific developmental regulator. In addition to its gene-regulatory function through DNA binding properties, WT-1 also regulates transcription by formation of protein-protein complexes. These properties place WT-1 as a major regulator of cell growth and differentiation. In view of these observations, we studied WT1 mRNA and protein in human endometrial extracts and in endometrial stromal cells (ESCs) differentiating into decidual cells in vitro, by RT-PCR and Western blotting, respectively. WT1 protein expression was also studied in situ in the proliferative and the secretory phase of the menstrual cycle in the early pregnant state. Analysis by PCR of total RNA prepared from human ESCs demonstrated the presence of WT1 mRNA and four WT1 mRNA splice variants. Western blot analysis of nuclear protein extracts from ESCs yielded one immunoreactive protein of the expected size (approximately 52–54 kDa) recognized by the WT1 antibody. Immunohistochemical staining showed that WT1 protein is localized only to nuclei of human endometrial stromal cells. It remains constant in the proliferative and the secretory phase of the menstrual cycle and is increased remarkably during decidualization in early pregnancy. ESCs decidualized in vitro were investigated for WT-1 expression, which confirmed that decidualizing stimuli (E2, medroxy-progesterone-acetate, and relaxin for 12 d or cAMP and progesterone for 1–4 d) induced WT-1 mRNA (P &lt; 0.05) and increased protein levels (P &lt; 0.05). These data indicate that in humans the WT1 gene is expressed in ESCs and its mRNA and protein levels remain constant in the proliferative and the secretory phase of the menstrual cycle and that WT1 mRNA and protein expression increases significantly in ESCs when these cells differentiate into decidual cells.

scholarly journals Up-regulation of Per1 expression by estradiol and progesterone in the rat uterus

2007 ◽  
Vol 194 (3) ◽  
pp. 511-519 ◽  
Author(s):  
Pei-Jian He ◽  
Masami Hirata ◽  
Nobuhiko Yamauchi ◽  
Masa-aki Hattori
Keyword(s):  
Stromal Cells ◽  
Clock Genes ◽  
Staining Intensity ◽  
Reproductive Organ ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Rat Uterus ◽  
Ovarian Steroids ◽  
Direct Regulation

It has been established that estrogen can alter circadian rhythms in behavior and endocrine physiology in rodents. The uterus is a reproductive organ that is critically dependent on regulation by ovarian steroids. Here, we examined the expression of Per1 in different compartments of the uterus, and explored whether the ovarian steroids could regulate Per1 expression employing ovariectomized rat uterus. RT-PCR analysis showed that Per1 was cyclically expressed in the uterus. As revealed by in situ hybridization, the staining intensity of Per1 mRNA was stronger at ZT 8 than at ZT 0 in the uterine luminal epithelium (LE), stroma (S), and myometrium (M) compartments, but was not changed in the glandular epithelium (GE). Both in situ hybridization and immunofluorescence analyses revealed that estradiol (E2) administration induced high expression of Per1 in the LE, GE, and M, and less expression in the S compartment. Progesterone (P4) treatment resulted in an obvious enhancement of Per1 expression in the LE, GE, and S, but unchanged in the M compartment. Furthermore, the E2- and P4-activated Per1 expression was significantly repressed by their respective antagonists, ICI182 780 and RU486. These findings were further supported by RT-PCR analysis of Per1 expression in cultured uterine stromal cells. Collectively, the present data indicate that E2 and P4 might be involved in modification of circadian rhythm via direct regulation of the expression of clock genes.

scholarly journals The Role of Decidual Subpopulations in Implantation, Menstruation and Miscarriage

2021 ◽  
Vol 3 ◽  
Author(s):  
Joanne Muter ◽  
Chow-Seng Kong ◽  
Jan J. Brosens
Keyword(s):  
Stromal Cells ◽  
Acute Stress ◽  
Embryo Implantation ◽  
Recurrence Risk ◽  
Tissue Remodelling ◽  
Endometrial Stromal Cells ◽  
Decidual Cells ◽  
Unk Cells ◽  
Acute Stress Response

In each menstrual cycle, the endometrium becomes receptive to embryo implantation while preparing for tissue breakdown and repair. Both pregnancy and menstruation are dependent on spontaneous decidualization of endometrial stromal cells, a progesterone-dependent process that follows rapid, oestrogen-dependent proliferation. During the implantation window, stromal cells mount an acute stress response, which leads to the emergence of functionally distinct decidual subsets, reflecting the level of replication stress incurred during the preceding proliferative phase. Progesterone-dependent, anti-inflammatory decidual cells (DeC) form a robust matrix that accommodates the conceptus whereas pro-inflammatory, progesterone-resistant stressed and senescent decidual cells (senDeC) control tissue remodelling and breakdown. To execute these functions, each decidual subset engages innate immune cells: DeC partner with uterine natural killer (uNK) cells to eliminate senDeC, while senDeC co-opt neutrophils and macrophages to assist with tissue breakdown and repair. Thus, successful transformation of cycling endometrium into the decidua of pregnancy not only requires continuous progesterone signalling but dominance of DeC over senDeC, aided by recruitment and differentiation of circulating NK cells and bone marrow-derived decidual progenitors. We discuss how the frequency of cycles resulting in imbalanced decidual subpopulations may determine the recurrence risk of miscarriage and highlight emerging therapeutic strategies.

scholarly journals 254.Interleukin-11 enhances endometrial stromal cell decidualisation via activation and inhibition of target genes

2004 ◽  
Vol 16 (9) ◽  
pp. 254
Author(s):  
C. A. White ◽  
E. Dimitriadis ◽  
A. Sharkey ◽  
C. J. Stoikos ◽  
L. A. Salamonsen
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Stromal Cells ◽  
Target Genes ◽  
Fold Change ◽  
Rt Pcr ◽  
Endometrial Stromal Cells ◽  
Cytoplasmic Staining ◽  
Decidual Cells ◽  
Custom Made

Differentiation of endometrial stromal cells into decidual cells is essential for successful embryo implantation. Interleukin (IL)-11 signalling is required for decidualisation in the mouse (1,2) and the expression pattern of IL-11 and its receptors during the menstrual cycle suggests a role for IL-11 in human decidualisation (3). Exogenous IL-11 has been shown to enhance hormone-induced decidualisation of human endometrial stromal cells in culture (4). This study aimed to determine the effects of IL-11 on downstream gene expression in endometrial stromal cells following 12 days of progesterone-induced decidualisation, and to examine the expression and functional significance of IL-11 target genes during this process. Stromal cells isolated from endometrial biopsies (n = 6) were decidualised with 17β-oestradiol and medroxyprogesterone acetate (EP) or EP with 100 ng/mL recombinant human IL-11. Medium was changed every 48 h, and total RNA extracted on Day 12 for gene expression analysis using custom-made 15K cDNA microarrays. Quantitative real-time RT-PCR was performed on the same samples to confirm gene expression levels. In subsequent experiments (n = 2), cells were cytocentrifuged onto glass slides for immunocytochemistry using specific antibodies. Microarray analysis revealed 16 upregulated and 11 downregulated cDNAs in EP + IL-11 compared to EP treated cells. Among these were IL-1β (6.1-fold upregulated) and insulin-like growth factor binding protein (IGFBP)-5 (3.6-fold downregulated). Using real-time RT-PCR, IL-11 was confirmed to increase IL-1β (fold change 1.3–107.1) and decrease IGFBP-5 (fold change 2.8–469.0) transcript abundance in 6 patients. Immunolocalisation of IL-1β in EP and EP + IL-11 treated cells revealed more intense vesicular cytoplasmic staining with IL-11 treatment, while staining intensity for IGFBP-5 was not affected. Interactions between IL-11 and its downstream targets IL-1β and IGFBP-5 are likely to have functional importance in early pregnancy, and may provide novel targets for the manipulation of human fertility. (1) Robb L, Li R, Hartley L, Nandurkar HH, Koentgen F, Begley CG (1998) Nat. Med. 4, 303–308. (2) Bilinski P, Roopenian D, Gossler A (1998) Gene Dev. 12, 2234–2243. (3) Dimitriadis E, Salamonsen LA, Robb L (2000) Mol. Hum. Reprod. 6, 907–914. (4) Dimitriadis E, Robb L, Salamonsen LA (2002) Mol. Hum. Reprod. 8, 636–643.

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