Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium

Matrix metalloproteinases (MMP) have specific spatial and temporal expression patterns in human endometrium and are critical for menstruation. Expression and activation mechanisms for proMMP-2 differ from other MMPs; in many cells proMMP-2 is specifically activated by membrane-type (MT)-MMPs. We examined the expression and localization of proMMP-2, MT1-MMP, and MT2-MMP in human endometrium across the menstrual cycle; and we examined the expression of MT1-MMP and activation of proMMP-2 in cultured endometrial stromal cells and their regulation by progesterone. MMP-2 was immunolocalized in 25 of 32 endometrial samples in all cellular compartments but with greatest intensity in degrading menstrual tissue. MT1-MMP mRNA was present throughout the cycle, and immunoreactive protein was detected in 24 of 32 samples, with the strongest staining in subsets of macrophages, neutrophils, and granular lymphocytes (but not mast cells or eosinophils) during the menstrual, mid-proliferative and mid-secretory phases. Patchy epithelial staining and staining of decidual cells, often periglandular in menstrual tissue, were also seen. MT2-MMP was more widespread than MT1-MMP without apparent cyclical variation and with maximal intensity in glandular epithelium. Cultured endometrial stromal cells released proMMP-2, and progesterone treatment significantly reduced the percentage level of its active (62 kDa) form (22.5 +/- 1.8% vs. 3.0 +/- 1.3%, without and with treatment, respectively, mean +/- SEM, P < 0.0001). This activation was blocked by a specific MMP inhibitor and restored following inhibitor removal. Progesterone also attenuated cell expression of MT1-MMP mRNA. We postulate that MT1-MMP activates proMMP-2 in endometrium, this activity being increased at the end of the cycle when progesterone levels fall, thus contributing to menstruation.     

NAD-dependent 15-hydroxyprostaglandin dehydrogenase activity in human endometrium

The specific activity of NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase was measured in human endometrial tissue obtained from ovulatory and anovulatory women. Employing PGE₂ as substrate, the specific activity of this enzyme was found to be highest in endometrial tissue during the secretory phase of the cycle (ovarian cycle days 15–25) and lowest in menstrual (days 1–5) and premenstrual (days 26–28) endometrium. The specific activity of prostaglandin dehydrogenase in endometrium of anovulatory women was low, being similar to that found in proliferative endometrium (days 6–14) of ovulatory women. Prostaglandin dehydrogenase activity was found in the cytosolic fraction prepared from endometrial tissue, and was found principally in the glandular epithelium following separation of endometrial glands and stromal cells.     

Paracrine regulation of endometrial function: interaction between progesterone and corticotropin-releasing factor (CRF) and activin A

Under the influence of ovarian steroid hormones, endometrial cells aer able to produce a wide variety of growth factors and peptide hormones that area believed to promote: (1) physiological growth and differentiation during the endometrial cycle; (2) decidualization, an essential preparative event for establishment of pregnancy; and (3) pathological growth and differentiation in endometriosis and cancer. Among the local factors produced by the human endometrium, corticotropin-releasing factor (CRF) and activin A have been evaluated in terms of localization and effects. CRF is a neuropeptide expressed by the epithelial and stromal cells of the human endometrium in increasing amounts from the endometrial proliferative to the secretory phase. CRF expression also increases in the pregnant endometrium, from early in the pregnancy until term. CRF-type 1 receptor mRNA is only expressed by stromal cells. Progesterone induces CRF gene expression and release from decidualized cells and CRF decidualizes cultured stromal endometrial cells. Urocortin, a CRF-related peptide, has been identified in endometrial epithelial and stromal cells, and its function is still under investigation. Activin A is a growth factor expressed in increasing amounts throughout endometrial phases by both epithelial and stromal cells. This growth factor is secreted into the uterine cavity with higher levels in the secretory phase. Maternal decidua expresses activin A mRNA in increasing amounts from early pregnancy until term. Human endometrium also expresses activin-A receptors and follistatin, its binding protein. Activin A decidualizes cultured human endometrial stromal cells (an effect reversed by follistatin) and modulates embryonic trophoblast differentiation and adhesion. Activin A is expressed in endometriosis and endometrial adenocarcinoma.     

Human Endometrial Side Population Cells Exhibit Genotypic,Phenotypic and Functional Features of Somatic Stem Cells

During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis for women''s cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC) population. Here we explore the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC.     

Gonadal steroids regulate the expression of aggrecanases in human endometrial stromal cells in vitro

The human endometrium undergoes cyclic change during each menstrual cycle in response to gonadal steroids. Proteolysis of endometrial extracellular matrix (ECM) is necessary to prepare this dynamic tissue for pregnancy. Proteolytic enzymes such as matrix metalloproteinase (MMP) and closely related a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) have been assigned key roles in the highly regulated cyclic remodelling of the endometrial ECM. We have previously shown that ADAMTS‐1 undergoes spatiotemporal changes in human endometrial stromal cells under the regulation of gonadal steroids. This suggests that other ADAMTS subtypes, known as aggrecanases, may contribute to the ECM remodelling events that occur in female physiological cycles and in preparation for pregnancy. To determine whether progesterone (P4), 17β‐estradiol (E2), or dihydrotestosterone (DHT), alone or in combination, are capable of regulating ADAMTS‐4, ‐5, ‐8 or ‐9 expression in human endometrial stromal cells in vitro. Real‐time quantitative PCR and Western blot analysis were used to measure ADAMTSs mRNA and protein levels in primary cultures of human endometrial stromal cells (n = 12). P4, DHT but not E2 have regulatory effects on ADAMTS‐8, ‐9 and ‐5 expression. Combined treatment with gonadal steroids did not show any synergistic or antagonistic effects. However, the synthetic steroid antagonists RU486 and hydroxyflutamide specifically inhibited the P4‐ or DHT‐mediated regulatory effects on ADAMTS expression. These studies provide evidence that the regulation of aggrecanases by gonadal steroids in human endometrial stromal cells may play an important role during decidualization.     

Regulation of FAS ligand expression by chemokine ligand 2 in human endometrial cells

Human endometrium is a dynamic tissue under the influence of numerous hormones, growth factors, and cytokines interacting to maintain a balance of cellular growth, differentiation, and apoptosis. We have previously demonstrated that several factors including interleukin-8, extracellular matrix, and steroid hormones modulate FASLG, one of the apoptotic molecules, in human endometrium. Chemokine ligand 2 (CCL2), a monocyte chemoattractant and activating factor, is a cytokine involved in endometrial function. CCL2 is elevated in the peritoneal fluid of women with endometriosis. We hypothesize that increased levels of CCL2 in the endometriotic environment may upregulate FASLG expression in human endometrial stromal cells and induce a local immunotolerance in endometriosis. To test our hypothesis, we studied the in vitro regulation of FASLG expression and apoptosis by CCL2 in endometrial stromal cells. Western blot analysis revealed that CCL2 upregulated FASLG protein expression in cultured endometrial stromal cells. Based on semiquantitative RT-PCR analysis, CCL2 did not alter either FAS or FASLG mRNA expression in endometrial stromal cells. Immunocytochemistry results from the same cells treated with CCL2 demonstrated upregulation of FASLG protein expression. CCL2 did not change rate of apoptosis in endometrial stromal cells as evaluated by TUNEL assay. However, an increased apoptotic rate was detected in Jurkat (T lymphocytes) cells cocultured with endometrial stromal cells previously treated with CCL2. We speculate that increased FASLG expression by CCL2 may induce apoptosis of T lymphocytes and thus produce an immunotolerant environment for the development of ectopic implants.     

Characterization and phenotypic variation with passage number of cultured human endometrial adenocarcinoma cells

Despite numerous endometrial cancer cell lines, little is know about the progression and transition of primary cultured endometrial tumours. Herein, a stage I grade III endometrial adenocarcinoma was maintained in primary culture and the phenotypic and protein expression changes were observed in relation to passage number. At early passage numbers, cultured human endometrial cancer (CHEC) cells displayed classic epithelial cell morphology, growing in groups in a glandular structure and staining positive for cytokeratin. However, with increasing passage number, CHEC cells changed in morphology to display a stromal phenotype which was accompanied by a significant reduction in cytokeratin and increases in alpha-actin and vimentin expression. Simultaneous culture of stromal cells isolated from the original tumour failed to show the same morphological characteristics or protein expression patterns. We further characterised CHEC cells through a screening of cancer related proteins, among others, caveolin-1 and Tissue factor in comparison with established cancer cell lines and corresponding non-cancerous cells. This report demonstrates that endometrial adenocarcinoma cells in culture can undergo phenotypic and protein expression changes reminiscent of epithelial-mesenchymal transition. This work suggests that primary tumours and cell lines displaying stromal morphologies may have undergone epithelial-mesenchymal transition from an adenocarcinoma origin.     

Regulation of gene expression and cellular localization of prostaglandin synthase by oestrogen and progesterone in the ovine uterus

Expression of the gene for prostaglandin synthase (PGS) was examined in whole endometrial tissue derived from ewes during the oestrous cycle (Days 4-14), on Day 15 of pregnancy and following ovariectomy and treatment with ovarian steroid hormones. Whilst no significant differences were seen in PGS mRNA concentrations analysed by Northern blot analysis in endometrial tissue during the oestrous cycle or in early pregnancy, treatment of ovariectomized (OVX) ewes with oestradiol-17 beta markedly reduced endometrial PGS mRNA concentration. There was no difference in PGS mRNA concentration in ewes treated with progesterone, either alone or in conjunction with oestrogen, from that in OVX controls. In contrast, differences in immunolocalization of PGS observed in uterine tissue from OVX-steroid-treated ewes were much more marked and reflected similar changes seen previously in the immunocytochemical distribution of endometrial PGS during the oestrous cycle. In OVX ewes and those treated with oestrogen, immunocytochemical staining for PGS was seen in stromal cells, but little immunoreactive PGS was located in the endometrial epithelial cells. However, in ewes treated with progesterone alone or with oestrogen plus progesterone, PGS was found in luminal and glandular epithelial cells and in stromal cells. Intensity of immunostaining for PGS in endothelial cells and myometrium did not differ between the treatments. Thus, whilst oestrogen lowers PGS mRNA in the endometrium, presumably in stroma, it may also increase the stability of the enzyme itself in the stromal cells. Although oestradiol-17 beta has no effect on PGS in endometrial epithelium, progesterone stimulates the production of PGS in endometrial epithelial cells without altering the overall abundance of PGS mRNA in the endometrium as a whole. Conceptus-induced changes in PGF-2 alpha release by ovine endometrium would not appear to be mediated via effects on PGS gene expression or protein synthesis.     

Interaction between insulin and androgen signalling in decidualization,cell migration and trophoblast invasion in vitro

Finely tuned decidualization of endometrial stromal fibroblasts into decidual cells is crucial for successful implantation and a healthy pregnancy. Both insulin and androgens are known to modulate decidualization, however, their complex effect on this process has not been fully elucidated. As hyperinsulinemia and hyperandrogenism are associated in clinical conditions, we aimed to investigate the interaction between insulin and androgens on decidualization. Primary human endometrial stromal cells were decidualized in vitro in the presence of insulin and/or androgens (dihydrotestosterone (DHT), testosterone). Gene or protein expressions of decidualization markers were measured, and cells size characteristics were determined. Migration of decidualizing endometrial stromal cells and invasion of HTR-8/SVneo trophoblast spheroids were assessed. We found that insulin and androgens in combination enhanced the upregulation of several decidualization markers including prolactin, tissue factor, tissue inhibitor of matrix metalloproteinase 3 and connexin-43, and also interacted in modulating cell size characteristics resulting in enlarged decidualizing cells. However, insulin and DHT together restricted the migration of decidualizing cells and invasion of trophoblast spheroids. Our findings suggest that insulin and androgens interact to potentiate the process of decidualization. On the other hand, inhibited cell migration and trophoblast invasion might negatively impact the function of decidualizing endometrial stromal cells.     

Clonogenicity of human endometrial epithelial and stromal cells

The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300-500/cm(2)) in serum medium or in serum- free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGF alpha, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except alpha(6)-integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.     

Characterization of an insulin-like growth factor binding protein, analogous to human pregnancy-associated secreted endometrial alpha 1-globulin, in decidua of the baboon (Papio anubis) placenta

The major secreted protein of the human decidua (pregnancy-associated endometrial alpha 1-globulin [alpha 1-PEG]), is an insulin-like growth factor-binding protein (IGF-BP) that is immunologically and biochemically similar to placental protein 12 (PP12) extracted from term human placenta. Since previous studies have demonstrated that the baboon and human endometrium synthesize and release a number of biochemically and immunologically related polypeptides in culture, this study was undertaken to further characterize a related IGF-BP in baboon placental tissues. Decidua, chorio-amniotic membranes with adhering decidua (CAM-D), and placental villi were obtained from pregnant baboons between Days 134 and 160 of gestation by Cesarean sections. Portions of tissue were either cultured in the presence of 35S-methionine, fixed for immunocytochemistry, or frozen in liquid nitrogen for cytosol extraction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of tissue culture media (TCM) revealed that the major secretory product of the decidua and CAM-D was an acidic polypeptide (Mr 33,000). Western blot analysis and immunoprecipitation of TCM with murine monoclonal antibody (B2H10) against human alpha 1-PEG demonstrated that this molecule, secreted by the baboon decidua and CAM-D, but not the placental villi, was immunologically identical to the human IGF-BP. Immunocytochemical localization of IGF-BP was intense in the cytoplasm of stromal cells in decidua and CAM-D and absent in the placenta. Gel filtration of TCM and cytosol followed by screening of eluates for 125I-IGF-I binding resolved two peaks (Mr greater than 100,000 and 35,000) of specific IGF-BP in decidua and CAM-D. The 35,000 peak had 100-200 times the binding capacity of the Mr greater than 100,000 peak and a Kd of 1.14-1.83 nM. The eluates contained in the Mr 35,000 peak were also immunoreactive to alpha 1-PEG, as accessed by a polyclonal radioimmunoassay. Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-PAGE revealed an immunoreactive complex of Mr 36,000, confirming that the baboon protein represents a high affinity IGF-BP. These studies indicate that the hypertrophied stromal cells of the baboon decidua and CAM-D synthesize and release an IGF-BP as their major secretory product, analogous to the situation in humans. The results of this study suggest that this protein may play a role in the regulation of IGF action during pregnancy.     

Epidermal growth factor inhibits 8-Br-cAMP-induced decidualization of human endometrial stromal cells

The effects of epidermal growth factor (EGF) on human endometrial stromal cells have not been characterized well, although production of EGF in endometrial epithelial and stromal cells and expression of EGF receptors in endometrial stromal cells have been reported. We investigated the effects of EGF on endometrial cell viability, 8-Br-cAMP-induced stromal decidualization, and prolactin secretion from decidualized endometrial stromal cells using an in vitro decidualization activity assay of human endometrial stromal cells. EGF did not show any significant effects on viable cell numbers of nondecidualized and 8-Br-cAMP-induced decidualized cells. Prolactin release from the 8-Br-cAMP-induced decidualized cells was not affected by EGF. However, EGF dose-dependently inhibited prolactin release from the stromal cells that were in the process of decidualization by co-stimulation with 8-Br-cAMP and EGF, though there was no significant change in viable cell numbers of the 8-Br-cAMP-stimulated decidualizing cells. Flow cytometric analysis revealed that 8-Br-cAMP enhanced EGF receptor expression on the endometrial stromal cells. These results indicate that endometrial EGF inhibits decidualization through autocrine/paracrine mechanisms.     

Stem Cell-Like Differentiation Potentials of Endometrial Side Population Cells as Revealed by a Newly Developed In Vivo Endometrial Stem Cell Assay

Background

Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman''s reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay.

Methodology/Principal Findings

ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells.

Conclusions/Significance

We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue reconstitution. Using this assay, we demonstrated that ESP cells differentiated into multiple endometrial lineages in the niche provided by whole endometrial cells, indicating that ESP cells are genuine endometrial stem/progenitor cells.     

Human endometrial stromal cells and decidual cells express cluster of differentiation (CD) 13 antigen/aminopeptidase N and CD10 antigen/neutral endopeptidase.

With specific monoclonal antibodies, we found that human endometrial stromal cells and decidual cells express two function-related surface antigens. Indirect immunofluorescence staining revealed that both endometrial stromal cells and decidual cells during the first trimester of pregnancy expressed cluster of differentiation (CD) 13 antigen and CD10 antigen, which are identical to aminopeptidase N and neutral endopeptidase, respectively. By flow cytometric analysis, CD13 antigen was detected on 82-93% of the examined cells, and CD10 antigen was detected on 75-93% of the examined cells in endometrial stromal cell-enriched preparations. Furthermore, peptidase activity was detected in these cell preparations by an assay based on the hydrolysis of alanine-p-nitroanilide into p-nitroaniline and alanine.