Regulation of Estrogen Activity in Human Endometrium: Effect of Cytokines on the Expression of Steroid Sulfatase and Sulfokinase mRNA in Human Endometrial Stromal Cells

The human endometrium has an immense regenerative capacity. Previously we identified a small population of clonogenic endometrial stromal cells or mesenchymal stem cells (MSC).1 Prospective isolation of MSC allows for their characterisation. We hypothesise that the expression of MSC marker, CD146, and pericyte/fibroblast marker, platelet-derived growth factor receptor-β (PDGFRβ), will enable the prospective isolation of endometrial MSCs. The aims of this study were to (1) determine if CD146 and PDGFRβ will prospectively isolate endometrial MSCs with clonogenic activity, (2) identify their location in human endometrium, and (3) determine the differentiation capacity of CD146+PDGFRβ+ stromal cells. Endometrial tissue from 13 ovulating women undergoing hysterectomy was digested with collagenase to produce single cell suspensions. Leukocytes and epithelial cells were removed. Stromal cells were analysed by flow cytometry, FACS sorted into enriched and depleted populations, and cultured for clonal analysis.1 Immunohistochemistry was performed on full thickness human endometrium. Sorted populations of stromal cells were passaged for culture in various differentiation media, and analysed for adipogenic, myogenic, chondrogenic or osteogenic differentiation by histological stains and RT-PCR. A small, consistent population of CD146+ endometrial stromal cells was identified (7.8 ± 1.1%, n = 8). In contrast, PDGFRβ expression varied (34.1 ± 9.7%, n = 5), and 2.5% of cells were CD146+PDGFRβ+. Clonogenicity of CD146+ stromal cells was significantly higher than CD146- stromal cells, 2.5 ± 1.1% and 1.2 ± 0.6%, respectively (n = 6, P = 0.03). CD146+ stromal cells were located perivascularly, similar to bone marrow MSCs, whereas PDGFRβ weakly stained the stroma, with stronger staining observed around the blood vessels. CD146+ cells differentiated into adipocytes, smooth muscle cells, chondrocytes and osteoblasts. This study identified CD146 as a marker of clonogenic endometrial stromal cells, and supports the perivascular location of endometrial MSCs. It also demonstrated that CD146+ cells can differentiate into four mesenchymal lineages. These data suggest that CD146 can be used for the prospective isolation of endometrial MSCs, which may be further enriched by PDGFRβ co-expression. (1)Chan RW, Schwab KE and Gargett CE (2004) Biology of Reproduction 70, 1738.

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The transcription factor HAND2 up-regulates transcription of the IL15 gene in human endometrial stromal cells

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012753 ◽

2020 ◽

Vol 295 (28) ◽

pp. 9596-9605 ◽

Cited By ~ 2

Author(s):

Hiromi Murata ◽

Susumu Tanaka ◽

Tomoko Tsuzuki-Nakao ◽

Takeharu Kido ◽

Maiko Kakita-Kobayashi ◽

...

Keyword(s):

Transcription Factor ◽

Stromal Cells ◽

Human Endometrium ◽

Luciferase Reporter ◽

Upstream Region ◽

Human Escs ◽

Endometrial Stromal Cells ◽

Immune Factor ◽

Unk Cells ◽

Cyclic Changes

Cyclic changes of the human endometrium, such as proliferation, secretion, and decidualization, occur during regular menstrual cycles. Heart– and neural crest derivatives–expressed transcript 2 (HAND2) is a key transcription factor in progestin-induced decidualization of human endometrial stromal cells (ESCs). It has been suggested that HAND2 regulates interleukin 15 (IL15), a key immune factor required for the activation and survival of uterine natural killer (uNK) cells. Activated uNK cells can promote spiral artery remodeling and secrete cytokines to induce immunotolerance. To date, no studies have evaluated the transcription factors that regulate IL15 expression in human ESCs. In the present study, we examined whether HAND2 controls IL15 transcriptional regulation in human ESCs. Quantitative RT-PCR and histological analyses revealed that HAND2 and IL15 levels increase considerably in the secretory phase of human endometrium tissues. Results from ChIP-quantitative PCR suggested that HAND2 binds to a putative HAND2 motif, which we identified in the upstream region of the human IL15 gene through in silico analysis. Using a luciferase reporter assay, we found that the upstream region of the human IL15 gene up-regulates reporter gene activities in response to estradiol and a progestin representative (medroxyprogesterone) in ESCs. The upstream region of the human IL15 gene also exhibited increasing responsiveness to transfection with a HAND2 expression vector. Of note, deletion and substitution variants of the putative HAND2 motif in the upstream region of IL15 did not respond to HAND2 transfection. These findings confirm that HAND2 directly up-regulates human IL15 transcription in ESCs.

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Progesterone Enhances Interleukin-15 Production in Human Endometrial Stromal Cells in Vitro1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.85.12.7023 ◽

2000 ◽

Vol 85 (12) ◽

pp. 4765-4770 ◽

Cited By ~ 3

Author(s):

Hidetaka Okada ◽

Tatsuya Nakajima ◽

Mayumi Sanezumi ◽

Akiko Ikuta ◽

Katsuhiko Yasuda ◽

...

Keyword(s):

Mrna Expression ◽

Stromal Cells ◽

Hormonal Control ◽

Human Endometrium ◽

Enzyme Linked Immunosorbent Assay ◽

Mrna Levels ◽

Dependent Manner ◽

Endometrial Stromal Cells ◽

Potent Inducer ◽

Interleukin 15

Interleukin-15 (IL-15) is a novel cytokine that stimulates lymphocyte proliferation and migration via a trimeric receptor sharing the β andγ signal-transducing chains with the IL-2 receptor. It is suggested that IL-15 is involved in regulating the proliferation and differentiation of uterine natural killer cells. In the human endometrium, we have recently reported that IL-15 messenger ribonucleic acid (mRNA) levels significantly increased during the secretory phase compared with those during the proliferative phase. In this study we investigated whether the female sex steroids progesterone (P) and estradiol (E2) regulate IL-15 messenger RNA (mRNA) and the secretion in human endometrial stromal cells (ESC) in vitro. Northern blot analyses revealed a significant increase in IL-15 mRNA levels in ESC treated with P alone or E2 plus P compared with vehicle. Furthermore, P is a potent inducer of IL-15 mRNA expression in ESC in a dose-dependent manner. On the other hand, E2 alone did not increase IL-15 mRNA expression. By enzyme-linked immunosorbent assay, IL-15 protein secretion was stimulated by P and further enhanced by combined treatment with E2 and P, whereas E2 alone was ineffective. It is suggested that IL-15 is deeply involved in the hormonal control of the human endometrium by P and E2.

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Cigarette Smoke Extract Activates Hypoxia-Inducible Factors in a Reactive Oxygen Species-Dependent Manner in Stroma Cells from Human Endometrium

Antioxidants ◽

10.3390/antiox10010048 ◽

2021 ◽

Vol 10 (1) ◽

pp. 48

Author(s):

Naoko Kida ◽

Yoshiyuki Matsuo ◽

Yoshiko Hashimoto ◽

Kenichiro Nishi ◽

Tomoko Tsuzuki-Nakao ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Stromal Cells ◽

Hypoxia Inducible Factor ◽

Reactive Oxygen ◽

Protein Stabilization ◽

Human Endometrium ◽

Dependent Manner ◽

Oxygen Species ◽

Endometrial Stromal Cells ◽

Hypoxic Conditions

Cigarette smoking (CS) is a major contributing factor in the development of a large number of fatal and debilitating disorders, including degenerative diseases and cancers. Smoking and passive smoking also affect the establishment and maintenance of pregnancy. However, to the best of our knowledge, the effects of smoking on the human endometrium remain poorly understood. In this study, we investigated the regulatory mechanism underlying CS-induced hypoxia-inducible factor (HIF)-1α activation using primary human endometrial stromal cells and an immortalized cell line (KC02-44D). We found that the CS extract (CSE) increased reactive oxygen species levels and stimulated HIF-1α protein stabilization in endometrial stromal cells, and that CS-induced HIF-1α-dependent gene expression under non-hypoxic conditions in a concentration- and time-dependent manner. Additionally, we revealed the upregulated expression of a hypoxia-induced gene set following the CSE treatment, even under normoxic conditions. These results indicated that HIF-1α might play an important role in CS-exposure-induced cellular stress, inflammation, and endometrial remodeling.

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Identification of genes regulated by interleukin-1β in human endometrial stromal cells

Reproduction ◽

10.1530/rep.1.00688 ◽

2005 ◽

Vol 130 (5) ◽

pp. 721-729 ◽

Cited By ~ 55

Author(s):

Marco Rossi ◽

Andrew M Sharkey ◽

Paola Viganò ◽

Giovina Fiore ◽

Rob Furlong ◽

...

Keyword(s):

Gene Expression ◽

Stromal Cells ◽

Cell Signalling ◽

Human Endometrium ◽

Interleukin 1Β ◽

Cellular Functions ◽

Tissue Remodelling ◽

Endometrial Stromal Cells ◽

Immune Modulators ◽

Cycle Regulation

Interleukin-1β (IL-1b) is an important immune regulatory factor that in human endometrium plays a role in both menstruation and implantation in the event of pregnancy. It promotes inflammatory-like processes and also stimulates tissue remodelling. We present a cDNA microarray study documenting the major effects of IL-1β on gene expression in stromal cells from human endometrium. Endometrial stromal cells from five normal healthy women at the mid secretory phase were cultured with or without IL-1β at 50 and 500 pg/ml for 48 h. cDNA microarrays were used to compare the levels of gene expression in total RNA isolated from cells stimulated with IL-1β. These cDNA arrays were produced containing 15 164 sequence-verified clones, which included genes known to be important in angiogenesis, immune modulators, apoptosis, cell signalling, extra-cellular matrix (ECM) remodelling and cell cycle regulation. Genes which were regulated by IL-1β were identified by analysis of the microarray data using the Significance Analysis of Microarrays software package. Upregulated (n= 23) and downregulated (n= 6) different genes were observed, which changed at least 3-fold, at a false discovery rate of less than 2% (P< 0.02). Our results have identified genes regulated by IL-1β, which are involved in leukocyte recruitment, ECM remodelling and other cellular functions. Changes in three genes, IL-8, colony-stimulating factor 2 and aldoketo reductase family 1 member 1, which were upregulated by IL-1β, were verified using real-time PCR. Novel functions regulated by IL-1β in endometrium, including genes involved in free radical protection, and fatty acid metabolism were also identified. These results also provide new insights into the role of IL-1β in disorders of the endometrium, especially in implantation-related infertility and endometriosis, in which this cytokine plays a major role.

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The potential role of tissue factor in regulating haemostasis, thrombosis and angiogenesis in human endometrium

Reproductive Medicine Review ◽

10.1017/s0962279901000230 ◽

2001 ◽

Vol 9 (2) ◽

pp. 119-139

Author(s):

F Schatz ◽

G Krikun ◽

CJ Lockwood

Keyword(s):

Blood Vessels ◽

Stromal Cells ◽

Luteal Phase ◽

Morphological Changes ◽

Maternal Blood ◽

Human Endometrium ◽

Primate Species ◽

Endometrial Stromal Cells ◽

Decidual Cells ◽

Ectopic Pregnancies

Decidual cells prevent peri-implantational endometrical bleedingDuring the menstrual cycle, the concerted effects of estradiol (E2) and progesterone induce human endometrial stromal cells (HESCs) to undergo decidualization, the collection of morphological, proliferative and biochemical changes that transforms stromal cells to decidual cells. In the luteal phase, progesterone initiates E2-primed HESCs to decidualize around blood vessels. Under continued progesterone stimulation, decidualization spreads throughout the late luteal phase and gestational endometrium. Among species with a haemochorial placenta, human trophoblasts are the most intrinsically invasive and human endometrium undergoes the most extensive decidualization reaction. Implanting blastocyst-derived syncytiotrophoblasts breach endometrial blood vessels embedded in a matrix of decidual cells. This invasive process institutes the primordial uteroplacental circulation, and provides the embryo with oxygen and nutrients. That it risks bleeding is clear from the phenomenon of chemical pregnancy, in which trophoblast-derived human chorionic gonadotrophin (hCG) appears transiently in the maternal blood followed by localized decidual bleeding. After endovascular invasion by syncytiotrophoblasts, extravillous cytotrophoblasts penetrate uterine spiral arteries and initiate the morphological changes that lead to increased intervillous blood flow. The occurrence of decidual haemorrhage during this period is associated with spontaneous abortion, abruption and preterm birth. Ectopic pregnancies are most frequently complicated by haemorrhage in primate species lacking a true decidua, thus underscoring the importance of decidual cells in preventing uterine bleeding.

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Expression of Delta-Like Protein 4 in the Human Endometrium

Endocrinology ◽

10.1210/en.2007-0477 ◽

2007 ◽

Vol 149 (1) ◽

pp. 15-19 ◽

Cited By ~ 24

Author(s):

J. Mazella ◽

S. Liang ◽

L. Tseng

Keyword(s):

Endothelial Cells ◽

Medroxyprogesterone Acetate ◽

Stromal Cells ◽

Vascular System ◽

Notch Signaling Pathway ◽

Human Endometrium ◽

Glandular Epithelium ◽

Clear Correlation ◽

Endometrial Stromal Cells ◽

Endometrial Cells

Activation of Delta-Notch signaling pathway promotes the development of the vascular system in embryo, normal adult tissues, and cancerous lesions. Delta and Notch genes are known to be expressed in endothelial cells, and little is known of their expression beyond the vascular system. The purpose of this study was to investigate whether Delta gene would be expressed in cells of the uterine endometrium. In this study, we found that the human endometrial cells expressed one of the Delta ligands, Delta-like 4 protein (Dll4). Dll4 was expressed in human endometrium in a spatiotemporal fashion. Immunohistochemistry studies showed the cytoplasm as well as membrane staining with apical localization both in the luminal and glandular epithelium and moderate diffuse staining in the cytoplasm of the stromal cells. Western blot analysis showed that the size of the endometrial Dll4 was identical to that in the human umbilical endothelial cells. The expression of Dll4 mRNA in human endometrial cells was quantitatively determined by real-time PCR. Dll4 mRNA expressed in the glandular epithelium showed large variations, and it was significantly elevated in the mid and late proliferative and early secretory endometrium. Endometrial stromal cells contained less Dll4 mRNA and had no clear correlation with the menstrual cycle. The effect of hormones was studied in the primary culture of isolated glandular epithelial and stromal cells. In glandular cells, estradiol had little effect, and medroxyprogesterone acetate significantly reduced the mRNAs compared with that of control. Relaxin induced the Dll4 mRNA. In stromal cells, both estradiol and medroxyprogesterone acetate reduced the Dll4 mRNA. To our knowledge, this is the first report of the expression of Dll4 in the endometrium. We propose that endometrial Dll4 may enhance the development of the endometrial microvascular system and facilitate the implantation of blastocyst in a fertile cycle.

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Neutral Endopeptidase Expressed by Decidualized Stromal Cells Suppresses Akt Phosphorylation and Deoxyribonucleic Acid Synthesis Induced by Endothelin-1 in Human Endometrium

Endocrinology ◽

10.1210/en.2006-0172 ◽

2006 ◽

Vol 147 (11) ◽

pp. 5153-5159 ◽

Cited By ~ 18

Author(s):

Akira Iwase ◽

Hisao Ando ◽

Tetsuro Nagasaka ◽

Daijiro Shibata ◽

Toko Harata ◽

...

Keyword(s):

Signaling Pathways ◽

Stromal Cells ◽

Neutral Endopeptidase ◽

Human Endometrium ◽

Kinase Signaling ◽

Endometrial Stromal Cells ◽

Endometrial Cells ◽

Akt Phosphorylation ◽

Endothelin 1 ◽

Eta Receptor

Endothelin-1 (ET-1) in human endometrium has been proposed to have a potential paracrine role, for its receptors are also present within this tissue. In addition, the expression of ET-1 varies during the menstrual cycle, and therefore, ET-1 may be involved in the cyclic change of the human endometrium, such as proliferation and decidualization. However, neither the inactivation of ET-1 in the endometrium nor the paracrine effect of ET-1 on endometrial cells has been determined. We investigated the production of ET-1 and the presence of neutral endopeptidase (NEP), which cleaves and inactivates ET-1, in primary cultured human endometrial cells. We found primary cultured endometrial epithelial cells, not stromal cells, to be the major source of ET-1. Western blot analysis and RT-PCR demonstrated that NEP was predominantly expressed by endometrial stromal cells. We also demonstrated that ET-1 stimulated the phosphorylation of Akt and DNA synthesis in endometrial stromal cells via the ETA receptor and phospahtidylinositol-3 kinase signaling pathways. The effect of ET-1 was regulated by NEP expressed by stromal cells. We also found that conditioned medium containing ET-1 from endometrial epithelial cell culture stimulated phosphorylation of Akt via the ETA receptor. In conclusion, ET-1 has a paracrine effect of Akt phosphorylation and cell proliferation on endometrial stromal cells, which occurs via the ETA receptor and phospahtidylinositol-3 kinase signaling pathways, and is regulated by cell-surface NEP.

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