Expression and steroid hormone regulation of TETs and DNMTs in human endometrium.

DNA methyltransferases (DNMTs) and ten-eleven translocation proteins (TETs) facilitate methylation and hydroxymethylation of DNA, respectively. DNMTs are widely studied with conflicting results on their regulation in the endometrium. While the role of TETs in the endometrium remains relatively unexplored. Deregulated expression of TETs and DNMTs are associated with endometrial pathologies. The aim of this study is to characterize the temporal TET expression in endometrium and to determine the hormonal regulation of TETs in comparison to DNMTs. mRNA expressions were quantified by real-time PCR in endometrial tissues from cycling women and localization was determined by immunohistochemistry. Hormonal regulation was investigated in endometrial epithelial and stromal cell lines following a 24 and 48 h treatment cycle. TET1 and 3 mRNA expressions were significantly upregulated in the mid-secretory phase. TET protein expression was ubiquitous in endometrial epithelium throughout the menstrual cycle except during the late-secretory phase, while stromal staining was scattered. TET1 mRNA was significantly upregulated in response to estrogen in stromal cells. Transcriptions of all three TETs were induced in response to progesterone treatment in epithelial cells. Only DNMT3b in epithelial cells and DNMT1 in stromal cells were significantly upregulated upon 24-h estrogen exposure following a significant decrease of DNMT1 when treated with 24 h of estrogen and progesterone. This study suggests that TETs are expressed in a cell-specific, dynamic manner in the endometrium and are responsive to steroid hormones. Investigating the role of TETs individually and with respect to DNMTs, will help to elucidate gene regulatory mechanisms in endometrial biology and pathologies.

Decidualisation of human endometrial stromal cells is associated with increased expression and secretion of prorenin.

BACKGROUND: In pregnancy, the decidualised endometrium expresses high levels of prorenin and other genes of the renin-angiotensin system (RAS) pathway. In this study we aimed to determined if the RAS was present in endometrial stromal cells and if decidualisation upregulated the expression of prorenin, the prorenin receptor ((P)RR) and associated RAS pathways. Immortalised human endometrial stromal cells (HESCs) can be stimulated to decidualise by combined treatment with medroxyprogesterone acetate (MPA), 17ß-estradiol (E2) and cAMP (MPA-mix) or with 5-aza-2'-deoxycytidine (AZA), a global demethylating agent. METHODS: HESCs were incubated for 10 days with one of the following treatments: vehicle, MPA-mix, a combination of medroxyprogesterone acetate (MPA) and estradiol-17ß alone, or AZA. Messenger RNA abundance and protein levels of prorenin (REN), the (P)RR (ATP6AP2), angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AGTR1), vascular endothelial growth factor (VEGF), and plasminogen activator inhibitor-1 (PAI-1) were measured by real-time PCR and ELISA's, respectively. Promyelocytic zinc finger (PLZF) and phospho-inositol-3 kinase (PIK3R1) mRNA abundances were also measured. RESULTS: HESCs expressed the prorenin receptor (ATP6AP2), REN, AGT, ACE and low levels of AGTR1. MPA-mix and AZA stimulated expression of REN. Prorenin protein secretion was increased in MPA-mix treated HESCs. E2 + MPA had no effect on any RAS genes. MPA-mix treatment was associated with increased VEGF (VEGFA) and PAI-1 (SERPINE1) mRNA and VEGF protein. CONCLUSIONS: An endometrial prorenin receptor/renin angiotensin system is activated by decidualisation. Since (P)RR is abundant, the increase in prorenin secretion could have stimulated VEGF A and SERPINE1 expression via Ang II, as both ACE and AGTR1 are present, or by Ang II independent pathways. Activation of the RAS in human endometrium with decidualisation, through stimulation of VEGF expression and secretion, could be critical in establishing an adequate blood supply to the developing maternal placental vascular bed.

Effect of cyclic AMP and estrogen/progesterone on the transcription of DNA methyltransferases during the decidualization of human endometrial stromal cells.

Progesterone, estrogen and cyclic adenosine monophosphate (cAMP) together regulate the decidualization of human endometrial stromal cells in a time-dependent manner. The role of DNA methylation and the three active DNA methyltransferases (DNMTs) in the regulation of decidualization is gaining interest but the exact role of this epigenetic mechanism during decidualization is largely unknown. We aimed to understand the effect of the main regulators of decidualization on the expression of the DNMTs and in turn on the expression of steroid hormone receptors during the decidualization. We conducted a time-course analysis from 6 h to 10 days to examine the change in gene expression of the DNMTs and the steroid hormone receptors over time in response to estradiol, medroxy-progesterone acetate (MPA) and dibutyryl-cAMP (db-cAMP) in a human endometrial stromal cells (HESC) cell line. Only the combination treatment with MPA-mix (estradiol + MPA + db-cAMP) up-regulated ERα, PGR, progesterone receptor B (PRB) and androgen receptor at 24 h. Both decidualization pathways of db-cAMP and estradiol/MPA, independently and combined, consistently down-regulated DNMT3B mRNA expression from 6 h till 10 days, whereas DNMT1 and DNMT3A mRNA expression were down-regulated transiently. Forced expression of DNMT3B in HESC for 10 days attenuated IGFBP1 mRNA and protein expression; and forced expression of DNMT3B combined with MPA-mix treatment synergistically increased the expression of PRB at 24 h. The HESC morphology and proliferation remained unchanged in response to forced expression of DNMT3B. In conclusion, mRNA expression of the DNMTs during decidualization is dynamic, so that expression varies according to the cAMP or estradiol/MPA pathway treatments that regulate them in a time-dependent manner. Although forced expression of DNMT3B by itself is insufficient to inhibit decidualization, forced expression of DNMT3B in combination with MPA-mix synergistically up-regulated PRB, as well as attenuated the expression of IGFBP1, the decidualization marker.

The effect of DNA methylation inhibitor 5-Aza-2'-deoxycytidine on human endometrial stromal cells.

BACKGROUND: Decidualization, the differentiation of endometrial stromal cells is a crucial step for successful implantation of an embryo, development of the placenta and completion of pregnancy to term. Epigenetic mechanisms are thought to be strongly involved in the regulation of processes controlling implantation, placentation, organ formation and foetal growth. Recent studies suggest that decreased DNA methylation facilitates a receptive endometrium. Hence, the aim of this project was to compare the transcriptional profile changes induced by the inhibitor of DNA methylation, 5-Aza-2'-deoxycytidine (AZA) to the transcriptional changes that happen during decidualization. METHODS AND RESULTS: When DNA methylation was inhibited in a human endometrial stromal cell (HESC) line with AZA, it resulted in the fibroblast-like stromal cells being transformed into decidual-like morphology after 9 days. Expression of both prolactin and insulin-like growth factor binding protein-1, the two established decidualization marker genes, were minimally up-regulated by AZA after 10 days of treatment. In a microarray of a three-way experiment between AZA-treated and oestradiol/progestin/cAMP-treated [medroxy-progesterone acetate (MPA)-mix] HESC and the untreated controls, we detected more than 1000 common genes that had a significant difference of expression compared with the controls. AZA-treated cells in the microarray significantly expressed 76 genes in common with the MPA-mix treated cells, and AZA treatment also differentially regulated 148 genes independently to that of MPA-mix treatment. The MPA-mix regulated at least 36 genes in the cell adhesion, extracellular matrix remodelling and RhoGTPase cytoskeletal reorganization pathway; AZA regulated 19 of these genes in common and 15 other RhoGTPase pathway genes. CONCLUSION: AZA induced some decidualization-like responses of endometrial stromal cells independently of progestins or cAMP, possibly via the cytoskeletal reorganization pathway of the RhoGTPase family.

Endometrial Stromal and Epithelial Cells Exhibit Unique Aberrant Molecular Defects in Patients With Endometriosis.

CONTEXT: Endometriosis is a chronic inflammatory disease that causes pain and infertility in women of reproductive age. OBJECTIVE: To investigate the pathologic pathways in endometrial stromal and epithelial cells that contribute to the manifestation of endometriosis. DESIGN: In vitro cellular and molecular analyses of isolated eutopic endometrial stromal and epithelial cells. METHODS: Eutopic stromal and epithelial cells from endometriotic and normal patients were isolated by fluorescence-activated cell sorting for paired sibling RNA sequencing and microRNA microarray. Aberrant pathways were identified using ingenuity pathway analysis networks and confirmed with in vitro modulation of the affected pathways in stromal and epithelial cell cultures. RESULTS: Both stromal versus epithelial cell types and paired endometriotic versus normal samples exhibited distinct hierarchical clustering. Compared to normal samples, there were 151 and 215 differentially expressed genes in the endometriotic stromal and epithelial populations, respectively, and concomitantly 9 and 16 differentially expressed microRNAs. Overall, endometriotic stromal and epithelial cells revealed distinct defects. In endometriotic stromal cells, key decidualization genes Zinc finger E-box Binding protein 1 (ZEB1), Heart And Neural crest Derivatives expressed 2 (HAND2), WNT4, and Interleukin 15 (IL-15) were found to be downregulated and Periostin (POSTN) and Matrix Metallopeptidase 7 (MMP7) were upregulated. Specifically, ZEB1 was downregulated in stromal cells by aberrant elevation in miR-200b. In contrast, ZEB1 was found to be upregulated in endometriotic epithelial cells through associated upregulation of transforming growth factor ß1 (TGFß1), inducer of the TGFß1-Bone Morphogenetic Protein 2 (BMP2)-MMP2-Prostaglandin-endoperoxide Synthase 2 (COX2)-ZEB1 pathway, which activates epithelial-mesenchymal transition. CONCLUSION: Manifestation of endometriosis involves dysregulation of unique molecular pathways within the diseased endometrial stromal and epithelial cells in the endometrium. Targeting the cell type-specific defects may offer a novel approach to treating endometriosis.

DNA methyltransferases and TETs in the regulation of differentiation and invasiveness of extra-villous trophoblasts.

Specialized cell types of trophoblast cells form the placenta in which each cell type has particular properties of proliferation and invasion. The placenta sustains the growth of the fetus throughout pregnancy and any aberrant trophoblast differentiation or invasion potentially affects the future health of the child and adult. Recently, the field of epigenetics has been applied to understand differentiation of trophoblast lineages and embryonic stem cells (ESC), from fertilization of the oocyte onward. Each trophoblast cell-type has a distinctive epigenetic profile and we will concentrate on the epigenetic mechanism of DNA methyltransferases and TETs that regulate DNA methylation. Environmental factors affecting the mother potentially regulate the DNA methyltransferases in trophoblasts, and so do steroid hormones, cell cycle regulators, such as p53, and cytokines, especially interlukin-1ß. There are interesting questions of why trophoblast genomes are globally hypomethylated yet specific genes can be suppressed by hypermethylation (in general, tumor suppressor genes, such as E-cadherin) and how invasive cell-types are liable to have condensed chromatin, as in metastatic cancer cells. Future work will attempt to understand the interactive nature of all epigenetic mechanisms together and their effect on the complex biological system of trophoblast differentiation and invasion in normal as well as pathological conditions.

Cell cycle regulation of human endometrial stromal cells during decidualization.

OBJECTIVE: Differentiation of endometrial stromal cells into decidual cells is crucial for optimal endometrial receptivity. Data from our previous microarray study implied that expression of many cell cycle regulators are changed during decidualization and inhibition of DNA methylation in vitro. In this study, we hypothesized that both the classic progestin treatment and DNA methylation inhibition would inhibit stromal cell proliferation and cell cycle transition. METHODS: The human endometrial stromal cell line (HESC) was treated from 2 days to 18 days with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (AZA), a mixture of estradiol/progestin/cyclic adenosine monophosphate ([cAMP]; medroxy-progesterone acetate [MPA mix]) or both. Cell growth was measured by cell counting, cell cycle transition and apoptosis were analyzed by flow cytometry, expression of cell cycle regulators were analyzed by quantitative polymerase chain reaction (qPCR) and Western blotting, and change in DNA methylation profiles were detected by methylation-specific PCR. RESULTS: Both AZA and MPA mix inhibited the proliferation of HESC for at least 7 days. Treatment with MPA mix resulted in an early G0/G1 inhibition followed by G2/M phase inhibition at 18 days. In contrast, AZA treatment inhibited cell cycle progression at the G2/M phase throughout. The protein levels of p21(Cip1)and 14-3-3σ were increased with both AZA and MPA mix treatments without any change in the DNA methylation profiles of the genes. CONCLUSIONS: Our data imply that the decidualization of HESC is associated with cell cycle arrest at G0/G1 phase initially and G2/M phase at later stages. Our results also suggest that p53 pathway members play a role in the cell cycle regulation of endometrial stromal cells.