Normal Human Endometrial Stromal Cells Research Articles

The roles of tight junction protein cingulin in human endometrioid endometrial cancer

ABSTRACT The bicellular tight junction molecule cingulin (CGN) binds to microtubules in centrosomes. Furthermore, CGN contributes to the tricellular tight junction (tTJ) proteins lipolysis-stimulated lipoprotein receptor (LSR) and tricellulin (TRIC). CGN as well as LSR decreased during the malignancy of endometrioid endometrial cancer (EEC). Although tTJ protein LSR is involved in the malignancy of some cancers, including EEC, the role of CGN is unknown. In this study, we investigated the roles of CGN with tTJ proteins in human EEC cells by using the CGN-overexpressing EEC cell line Sawano. In 2D cultures, CGN was colocalized with LSR and TRIC at tTJ or at γ-tubulin-positive centrosomes. In immunoprecipitation with CGN antibodies, CGN directly bound to LSR, TRIC, and β-tubulin. Knockdown of CGN by the siRNA decreased the epithelial barrier and enhanced cell proliferation, migration and invasion, as well as knockdown of LSR. In the Sawano cells cocultured with normal human endometrial stromal cells, knockdown of CGN decreased expression of LSR and TRIC via MAPK and AMPK pathways. In 2.5D cultures, knockdown of CGN induced the formation of abnormal cysts and increased the permeability of FD-4 to the lumen. In 2D and 2.5D cultures, treatment with β-estradiol with or without EGF or TGF-β decreased CGN expression and the epithelial permeability barrier and enhanced cell migration, and pretreatment with EW7197+AG1478, U0126 or an anti-IL-6 antibody prevented this. In conclusion, CGN, with tTJ proteins might suppress the malignancy of human EEC and its complex proteins are sensitive to estrogen and growth factors derived from stromal cells.

Transcription Factor STAT3-Activated LDHB Promotes Tumor Properties of Endometrial Cancer Cells by Inducing MDH2 Expression.

The pathogenesis of endometrial cancer (EC) involves the regulation of lactate dehydrogenases. However, the role and mechanism of lactate dehydrogenase-B (LDHB) in EC progression have not been studied. The mRNA levels of LDHB and malate dehydrogenase 2 (MDH2) were detected by quantitative real-time polymerase chain reaction. Protein expression was checked by western blotting and immunohistochemistry assays. Cell proliferation, apoptosis, and invasion were analyzed by 5-Ethynyl-2'-deoxyuridine, transwell, and flow cytometry assay, respectively. Glycolysis was investigated using Glucose Assay Kit, CheKine™ Micro Lactate Assay Kit, and ADP/ATP ratio assay kit. An in vivo tumor formation assay was conducted to disclose the effect of LDHB on tumor growth in vivo. The associations among signal transducer and activator of transcription 3 (STAT3), LDHB, and MDH2 were predicted through JASPAR or GeneMANIA online database and identified by chromatin immunoprecipitation assay, dual-luciferase reporter assay, and co-immunoprecipitation assay. LDHB expression was increased in EC tissues and cells in comparison with normal endometrial tissues and human endometrial stromal cells. LDHB had the potential as a biomarker to predict the prognosis of EC patients. In addition, LDHB knockdown inhibited the proliferation, invasion, and glycolysis and promoted apoptosis of RL95-2 and Ishikawa cells. LDHB knockdown inhibited tumor property of Ishikawa cells in vivo. STAT3 bound to the promoter region of LDHB, and STAT3 silencing-induced effects were relieved after LDHB upregulation. LDHB interacted with and regulated MDH2 expression. Moreover, MDH2 overexpression rescued LDHB knockdown-induced effects on EC cell phenotypes. STAT3-activated LDHB promoted endometrial cancer cell malignancy by inducing MDH2 production.

Oleuropein suppresses endometriosis progression and improves the fertility of mice with endometriosis

BackgroundEndometriosis is an estrogen-dependent inflammatory reproductive disease. Therefore, systematic estrogen depletion and anti-inflammatory drugs are the current treatment for endometriosis. However, current endometriosis treatments have low efficacy and cause adverse effects in endometriosis patients. Consequently, alternative endometriosis treatments targeting endometriosis-specific factors are in demand. In this context, ERβ was selected as a druggable target for endometriosis due to its critical role in progression. Therefore, selective targeting of ERβ without inhibiting ERα activity would be a new paradigm for endometriosis treatment to overcome the low efficacy and adverse effects of hormonal endometriosis therapy.MethodsCell-based ERβ and ERα activity assay systems were employed to define a selective ERβ-inhibiting chemical product from a library of natural products. A surgically induced endometriosis mouse model was used to determine whether an ERβ inhibitory drug suppressed endometriosis progression. Mice with endometriosis were randomly separated and then orally treated with vehicle or 25 mg/kg oleuropein (once a day for 21 days), an ERβ inhibitory drug. The volume of endometriotic lesions or luciferase activity of endometriotic lesions was examined to define the growth of ectopic lesions in mice with endometriosis. The metabolite and levels of metabolic enzymes of the liver and kidney were determined in the serum of female mice treated with vehicle and oleuropein (25 mg/kg, once a day for 21 days) to define the toxicity of oleuropein. The in vitro decidualization assay was conducted with normal human endometrial stromal cells and endometriotic stromal cells to determine whether oleuropein overcomes decidualization in endometriosis patients. The pregnancy rate and pup numbers of C57BL/6 J female mice with endometriosis treated with vehicle or oleuropein (n = 10/group) were determined after mating with male mice. The cytokine profile in endometriotic lesions treated with vehicle and oleuropein (25 mg/kg) was determined with a Mouse Cytokine Array Kit.ResultsAmong natural products, oleuropein selectively inhibited ERβ but not ERα activity in vitro. Oleuropein treatment inhibited the nuclear localization of ERβ in human endometrial cells upon estradiol treatment. Oleuropein (25 mg/kg) treatment suppressed the growth of mouse (6.6-fold) and human (sixfold) ectopic lesions in mice with endometriosis compared to the vehicle by inhibiting proliferation and activating apoptosis in endometriotic lesions. Oleuropein treatment did not cause reproductive toxicity in female mice. Additionally, mice with endometriosis subjected to oleuropein treatment had a higher pregnancy rate (100%) than vehicle-treated mice (70%). Furthermore, oleuropein treatment partially recovered the decidualization impact of human endometriotic stromal cells from endometriotic lesions compared to the vehicle. Oleuropein-treated mice with endometriosis exhibited significantly lower levels of cytokines directly regulated by ERβ in ectopic lesions than vehicle-treated mice, illustrating the improvement in the hyperinflammatory state of mice with endometriosis.ConclusionsOleuropein is a promising and novel nutraceutical product for nonhormonal therapy of endometriosis because it selectively inhibits ERβ, but not ERα, to suppress endometriosis progression and improve the fertility of mice with endometriosis.

A small compound spindlactone A sensitizes human endometrial cancer cells to TRAIL-induced apoptosis via the inhibition of NAD(P)H dehydrogenase quinone 1.

IntroductionSpindlactone A (SPL-A) is a novel small molecule inhibitor of TACC3 that selectively inhibits the nucleation of centrosome microtubules and induces mitotic arrest in ovarian cancer cells. SPL-A is derived from dicoumarol which inhibits the activity of NAD(P)H dehydrogenase quinone oxidoreductase 1 (NQO1). This study aimed to investigate the mechanism by which SPL-A enhances TRAIL-induced apoptosis in endometrial carcinoma cells.Materials and methodsEndometrial carcinoma cells were treated with SPL-A and/or TRAIL, and the apoptosis and protein expression in the treated cells were examined.ResultsCombined treatment with SPL-A and TRAIL significantly induced apoptosis in various human endometrial carcinoma cells, but not in normal human endometrial stromal cells and endometrial epithelial cells. Notably, both NQO1 inhibitor ES936 and NQO1 siRNA enhanced TRAIL-induced apoptosis of endometrial carcinoma cells. Furthermore, SPL-A downregulated the expression of c-FLIP, Bcl-2, Bcl-xl, and Mcl-1, while increasing p53 expression.ConclusionIn particular, luciferase assay showed that SPL-A inhibited Bcl-2 promoter activity, and p53 inhibitor PFT-α could reverse the effect of SPL-A on Bcl-2 expression. Moreover, Bcl-2 overexpression inhibited the apoptosis induced by SPL-A and TRAIL. Taken together, our results suggest that SPL-A sensitizes endometrial cancer cells to TRAIL-induced apoptosis via the regulation of apoptosis-related proteins and the inhibition of NQO1 activity.

Adiponectin Effect on Nitric Oxide Secretion by Normal and Endometriotic Human Endometrial Stromal Cells: in vitro Study

La endometriosis es una enfermedad dependiente de estrogenos que se presenta en mujeres en edad reproductiva. La adiponectina y el oxido nitrico (ON) tienen un papel importante en las funciones fisiologicas, especialmente en el sistema reproductivo humano. Los niveles de ON aumentan en los pacientes con endometriosis, pero los niveles de adiponectina en suero disminuyen. El objetivo fue determinar el efecto de la adiponectina sobre la secrecion de ON por las celulas estromales de endometrio humano, tanto normales como con endometriosis, en medio de cultivo. En este estudio experimental, las celulas estromales de endometrio normales (n= 10) y las biopsias de endometrio con endometriosis (n= 10) se tomaron en condiciones de esterilidad. Las celulas estromales fueron aisladas y cultivadas en un medio DMEM/F12, y se sometieron a distintas concentraciones de adiponectina (0, 10, 100, y 200 ng/ml) durante 24 y 48 horas. El ensayo con ON se realizo a los sobrenadantes obtenidos por el metodo de Greiss. Los datos recolectados fueron analizados por ANOVA de una via y un valor p 0,05). La adiponectina tiene efectos sobre la secrecion de oxido nitrico por las celulas estromales endometriales humanas en cultivo.

Danazol regulates the functions of normal human endometrial stromal cell subpopulations by modifying endometrial cytokine networks

Local danazol therapy can improve endometriotic signs and symptoms without causing any menstrual disorders. As a consequence, certain direct actions of danazol on endometriotic tissues have been proposed, but the mechanisms of these actions have not been clarified. In the present study, the direct effects of danazol on normal human endometrial stromal cells (ESCs) were examined using in vitro decidualization assays. Danazol did not affect the viable cell numbers of unstimulated ESCs or 8Br-cAMP-stimulated decidualized ESCs, but significantly enhanced the viable cell numbers of 8-Br-cAMP-stimulated ESCs during decidualization in a dose-dependent manner. Danazol had no effect on PRL secretion by 8-Br-cAMP-stimulated decidualized ESCs. Danazol, as well as progesterone and medroxyprogesterone acetate (MPA), induced ESC decidualization. Danazol synergistically enhanced the differentiation process of 8-Br-cAMP-stimulated ESCs during decidualization. Although progesterone and MPA increased G-CSF and IL-8 secretion by ESCs in similar manner to 8-Br-cAMP, danazol had no such effects. Moreover, remarkable increases in G-CSF and IL-8 secretions by 8-Br-cAMP-stimulated ESCs during decidualization were completely inhibited by cotreatment with danazol. These results indicate that danazol has specific pharmacological effects on ESCs, rather than progesterone-like effects or similar effects to those reported for endometrial cytokines. According to the results, normal human ESCs can be classified into at least four functional subpopulations. Therefore, under certain circumstances, danazol has similar or opposite effects on ESCs to certain endometrial cytokines, suggesting that it regulates functional cellular subpopulation ratios of normal human ESCs by modifying the endometrial cytokine network in endometrial stromal tissues.

Modulation of vascular endothelial growth factor and thymidine phosphorylase in normal human endometrial stromal cells

To study the regulation of vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP), two angiogenic factors, in cultured endometrial stromal cells. The effects of steroids, E2, and P, the gonadotropin, hCG, and hypoxia were investigated. Quantitative experimental study. Academic medical department. Women undergoing hysterectomy for benign causes. Endometrial cells were collected from subjects and cultured. The secretion of VEGF in supernatant media and the numbers of cells expressing VEGF or TP. Estradiol increased VEGF secretion and the number of cells that contained VEGF and TP, and those effects were inhibited by hCG. Human chorionic gonadotropin alone could increase number of cells that expressed VEGF and TP. Hypoxia increased both VEGF secretion and number of cells containing VEGF and TP. Progesterone had no observed effect on VEGF secretion or number of VEGF- or TP-containing cells. Vascular endothelial growth factor and TP are present in stromal cells of normal endometrium, and E2 may interact with gonadotropins to regulate angiogenic compounds to modulate stromal functioning.

Leptin inhibits decidualization and enhances cell viability of normal human endometrial stromal cells

Recent reports have demonstrated that the peritoneal fluid and serum concentrations of leptin are increased in women with endometriosis. However, the pathophysiological roles of leptin in endometriosis have not been well characterized. In this study, we examined the direct effects of leptin on normal human endometrial stromal cells using an in vitro decidualization assay system with 8-Br-cAMP, a decidualization inducer. No effects of leptin on cell viability and prolactin secretion were found in unstimulated endometrial stromal cells. Leptin dose-dependently enhanced the viability of stromal cells co-stimulated with 8-Br-cAMP and leptin while PRL secretion from the cells was significantly inhibited in a dose-dependent manner. As for 8-Br-cAMP-stimulated cells, leptin significantly enhanced their cell viability in a dose-dependent manner but not their PRL secretion. These results indicate that leptin enhances the cell viability of PRL-non-secreting 8-Br-cAMP-stimulated stromal cells, and that it inhibits the decidualization process of endometrial stromal cells. Increased leptin in endometriotic patients might play an antiapoptotic role in some activated ESCs in the peritoneal cavity to stimulate endometrial cell implantation, and might cause infertility by inhibiting stromal decidualization.

Fas antigen (CD95) mediates cell survival signals to regulate functional cellular subpopulations in normal human endometrial stromal cells

Fas antigen (CD95) is expressed on almost all types of human cells and is believed to mediate receptor-specific apoptotic signals. In human endometrial tissues, high Fas and Fas ligand expressions and Fas-mediated apoptosis in endometrial epithelial cells have been discussed in many reports but no study has examined Fas-mediated signals in endometrial stromal cells. In this study we investigated Fas expression and Fas-mediated signals of normal human endometrial stromal cells. Flow cytometric analysis revealed Fas antigen expression on the stromal cells and their Fas expression was enhanced by 8-Br-cAMP, a strong inducer of decidualization. Neither short-term nor long-term cultures with anti-Fas IgM affected proliferation or viability of the stromal cells. Anti-Fas IgM alone affected neither viable cell numbers nor PRL release of unstimulated stromal cells. However, anti-Fas IgM dose-dependently stimulated viable cell numbers of stromal cells co-stimulated with 8-Br-cAMP and anti-Fas IgM, whereas PRL secretion of the co-stimulated cells was not affected. Anti-Fas IgM dose-dependently stimulated viable cell numbers of 8-Br-cAMP-stimulated cells but did not affect PRL secretion of 8-Br-cAMP-induced decidualized cells. These results indicate that Fas antigen on human endometrial stromal cells cannot mediate receptor-specific apoptotic signals, and that Fas-mediated signals stimulate survival of 8-Br-cAMP-stimulated non-decidualized stromal cells. Thus, stimulation of Fas-antigen on the endometrial stromal cells enhances anti-apoptotic/survival signals in certain stromal cells, autoregulating functional cellular subpopulations of human endometrial stromal cells in a paracrine manner.

Regulation of the cellular subpopulation ratios of normal human endometrial stromal cells by macrophage colony-stimulating factor

Normal human endometrial stromal cells (ESCs) stimulated with 8-Br-cAMP secrete significantly large amounts of PRL and granulocyte colony-stimulating factor (G-CSF), whereas unstimulated stromal cells secreted little. However, there is no relation between PRL and G-CSF levels secreted from the stimulated cells, which suggests that PRL-secreting stromal cells may not completely coincide with the G-CSF-producing stromal cells. Recently we found that macrophage colony-stimulating factor (M-CSF) inhibits 8-Br-cAMP-induced decidualization, differentiation to prolactin (PRL)-secreting cells, by suppressing viable decidualizing cells. Therefore, we investigated the effects of M-CSF on G-CSF secretion from normal human endometrial stromal cells using an in vitro decidualization activity assay. M-CSF did not show any significant effects on viable cell numbers of unstimulated ESCs, while M-CSF dose-dependently enhanced G-CSF release from the non-decidualized stromal cells. A high concentration of M-CSF strongly suppressed the viable cell numbers and PRL secretion of stromal cells co-stimulated with 8-Br-cAMP and M-CSF, although G-CSF release from the co-stimulated stromal cells was not affected by M-CSF. Moreover, M-CSF did not affect viable cell numbers, PRL secretion or G-CSF secretion of 8-Br-cAMP-stimulated cells. These results indicate that M-CSF enhances G-CSF secretion from 8-Br-cAMP-unstimulated human endometrial stromal cells but not from 8-Br-cAMP-stimulated stromal cells, thus suggesting that there exists a functional subpopulation of G-CSF-secreting stromal cells that are different from the predecidualized ESCs that differentiate into PRL-secreting ESCs under stimuli with 8-Br-cAMP. Hence, M-CSF may autoregulate functional cellular subpopulations of human endometrial stromal cells in an autocrine or a paracrine manner.

Oncostatin M inhibits decidualization of normal human endometrial stromal cells

It is well known that oncostatin M binds and activates leukemia inhibitory factor-specific receptors while leukemia inhibitory factor cannot bind oncostatin M-specific receptors. In this study, the differential effects of oncostatin M and leukemia inhibitory factor on cell survival and decidualization of normal human endometrial stromal cells were investigated by using 8-Br-cAMP-induced decidualization assay. Oncostatin M did not affect the viable cell numbers or the prolactin release of unstimulated stromal cells. However, oncostatin M dose-dependently suppressed prolactin releases from 8-Br-cAMP-stimulating stromal cells but enhanced their viable cell numbers. Although oncostatin M significantly enhanced viable cell numbers of 8-Br-cAMP-stimulated stromal cells, it did not affect prolactin secretion from 8-Br-cAMP-induced decidualized cells. Leukemia inhibitory factor significantly enhanced viable cell numbers of 8-Br-cAMP-stimulating cells in a dose-dependent manner as did oncostatin M, while leukemia inhibitory factor did not show any significant suppression of PRL secretion from 8-Br-cAMP-stimulating cells. These results indicate that oncostatin M inhibits the 8-Br-cAMP-induced decidualization process via oncostatin M-specific receptors and that oncostatin M and leukemia inhibitory factor enhance cell survival of 8-Br-cAMP-stimulated prolactin-non-secreting stromal cells mainly via leukemia inhibitory factor receptors. Thus, oncostatin M may autoregulate human endometrial stromal cell survival and decidualization in a paracrine manner.

Leukemia inhibitory factor regulates cell survival of normal human endometrial stromal cells

Leukemia inhibitory factor (LIF) is produced locally in decidual tissues, but its direct effects on endometrial stromal cells have not been well characterized. In this study, we examined the direct effects of LIF on normal human endometrial stromal cells using an in vitro decidualization assay system with 8-Br-cAMP, a decidualization inducer. We found no effects of LIF on cell viability and prolactin secretion of unstimulated endometrial stromal cells. LIF dose-dependently enhanced cell viability, but not prolactin secretion of 8-Br-cAMP-stimulated cells. LIF dose-dependently enhanced the viability of stromal cells co-stimulated with 8-Br-cAMP and LIF without any significant effect on PRL secretion from the cells. Further, the extracellular matrix did not affect these stimulatory effects of LIF on cell viability of 8-Br-cAMP-stimulated endometrial stromal cells. These results indicate that LIF enhances the cell viability of PRL-non-secreting 8-Br-cAMP-stimulated stromal cells, and that the cell survival signals generated by LIF are independent of those generated by the extracellular matrix. LIF produced locally in decidual tissues may enhance cell viability in certain activated endometrial stromal cells in an autocrine or paracrine manner, and possibly protect against cell damage during embryo implantation and trophoblastic invasion.

Interleukin-11 enhances cell survival of decidualized normal human endometrial stromal cells

High expression of interleukin-11 (IL-11) and the IL-11 receptor alpha chain in developing decidual cells in mice has been reported ,and mice lacking IL-11 receptor expression have been reported to show impaired implantation owing to defective decidualization. However ,the direct effect of IL-11 on endometrial stromal cells has not been studied. In this study ,we examined the direct effects of IL-11 on normal human endometrial stromal cells using an in vitro decidualization assay system. IL-11 enhanced cell viability and prolactin secretion of 8-Br-cAMP-induced decidualized cells but not of nonstimulated stromal cells. IL-11 dose-dependently enhanced the viability of stromal cells co-stimulated with 8-Br-cAMP and IL-11 without any significant effect on prolactin secretion from the cells. The extracellular matrix did not affect the effect of IL-11 on the viability of 8-Br-cAMP-stimulated stromal cells. These results indicate that IL-11 enhances the viability of 8-Br-cAMP-stimulated stromal cells and of decidualized stromal cells ,and that the cell survival signals generated by IL-11 are independent of those generated by the extracellular matrix. IL-11 produced locally in decidual tissues may enhance the viability of decidualized stromal cells and possibly protect against cell damage during embryo implantation and trophoblastic invasion.

Regulation of Human Endometrial Stromal Decidualization by Macrophage Colony-Stimulating Factor

Decidualized human endometrial stromal cells and human decidual CD56+ cells are reported to produce macrophage colony-stimulating factor (M-CSF), although its physiological roles remain unclear. In this study, the effects of M-CSF on cell viability and 8-Br-cAMP-stimulated decidualization of normal human endometrial stromal cells are examined by using an in vitro decidualization activity assay system. M-CSF alone affected neither the viable cell numbers nor PRL release of nonstimulated stromal cells, while high concentrations of M-CSF strongly suppressed the viable cell numbers and PRL secretion of stromal cells costimulated with 8-Br-cAMP and M-CSF. However, M-CSF did not suppress the viable cell numbers and PRL secretion of 8-Br-cAMP-induced decidualized cells. These results indicate that high concentrations of M-CSF can suppress the cell viability of stromal cells in the decidualization process, and inhibit decidualization of endometrial stromal cells. Thus, nonpregnant decidual M-CSF may autoregulate decidualization and the viable cell numbers of endometrial stromal cells in a paracrine manner.

Interleukin-11 enhances cell survival of decidualized normal human endometrial stromal cells

High expression of interleukin-11 (IL-11) and the IL-11 receptor alpha chain in developing decidual cells in mice has been reported ,and mice lacking IL-11 receptor expression have been reported to showimpaired implantation owing to defective decidualization. However ,the direct effect of IL-11 on endometrial stromal cells has not been studied. In this study ,we examined the direct effects of IL-11 onnormal human endometrial stromal cells using an in vitro decidualization assay system. IL-11 enhanced cell viability and prolactin secretion of 8-Br-cAMP-induced decidualized cells but not of nonstimulatedstromal cells. IL-11 dose-dependently enhanced the viability of stromal cells co-stimulated with 8-Br-cAMP and IL-11 without any significant effect on prolactin secretion from the cells. The extracellularmatrix did not affect the effect of IL-11 on the viability of 8-Br-cAMP-stimulated stromal cells.These results indicate that IL-11 enhances the viability of 8-Br-cAMP-stimulated stromal cells and ofdecidualized stromal cells ,and that the cell survival signals generated by IL-11 are independent of those generated by the extracellular matrix. IL-11 produced locally in decidual tissues may enhance theviability of decidualized stromal cells and possibly protect against cell damage during embryo implantation and trophoblastic invasion.

Immunohistochemical analysis of steroid receptors and glycodelin A (PP14) in isolated glandular epithelial cells of normal human endometrium.

Highly purified fractions of isolated endometrial cells can be useful for investigating endometrial function. After a first collagenase digestion, normal human endometrial stromal and epithelial cells were separated by filtration. Glands were purified further by two collagenase digestion steps, filtration, differential sedimentations, and Ficoll gradient centrifugation. Epithelial cells were polyhedral and grew as islands in a whorl-like wavy pattern around glandular fragments. High cell culture purity was confirmed with the positive immunohistochemical reaction against cytokeratin 7,8,18,19. Isolated human glands had a similar distribution pattern of estrogen receptor (ER) and progesterone receptor (PR) as observed in vivo, suggesting that glands have a functional hormone receptor system at the time of plating. Using a specific monoclonal antibody against glycodelin A (GdA), a characteristic cyclical expression was demonstrated during the menstrual cycle. The GdA reaction was weak in the proliferative phase, increasing significantly till the late secretory phase, suggesting a similar GdA concentration in vitro as observed in vivo glands. In conclusion, this method could be a model for studying endometrial glandular cells from different menstrual phases, endometrial cell interactions, implantation mechanisms, GdA regulation mechanisms, and pharmacological or other influences on ER and PR alteration.

Enhancement of human endometrial stromal decidualization by bestatin, an aminopeptidase-N inhibitor

We have re-evaluated the effects of bestatin, an aminopeptidase-N (CD13) inhibitor, on in vitro decidualization of normal human endometrial stromal cells by using an in vitro decidualization activity assay. Bestatin did not show any effects on the viability of both the decidualizing stromal cells co-stimulated with 8-Br-cAMP and bestatin and the 8-Br-cAMP-induced decidualized stromal cells, or on prolactin release from the decidualized stromal cells. However, bestatin dose-dependently enhanced prolactin release from the decidualizing stromal cells co-stimulated with 8-Br-cAMP and bestatin. These results indicate that bestatin enhances cAMP-mediated decidualization of human endometrial stromal cells and suggests that membrane aminopeptidase-N in the human endometrium is involved in the regulation of endometrial differentiation by inhibiting cAMP-mediated decidualization signals in endometrial stromal cells.

Production and physiological function of granulocyte colony-stimulating factor in non-pregnant human endometrial stromal cells

Effects of granulocyte colony-stimulating factor (G-CSF) on proliferation and differentiation of normal human endometrial stromal cells were investigated in an in vitro decidualization culture of stromal cells. Unstimulated stromal cells secreted little prolactin and G-CSF, whereas 8-Br-cAMP-stimulated stromal cells secreted higher levels. There was no relationship, however, between the levels of prolactin and G-CSF secreted from the stimulated cells. Detectable levels of prolactin secretion were not found in two of six stromal cell cultures stimulated with 8-Br-cAMP; however, these two culture supernatants contained high concentrations of G-CSF. Co-stimulation of the stromal cells with 8-Br-cAMP and G-CSF enhanced prolactin secretion from the stimulated cells in a G-CSF concentration-dependent manner without any change in viable cell numbers. However, G-CSF did not affect prolactin secretion or viable cell numbers of 8-Br-cAMP-stimulated decidualized cells. These results indicate that G-CSF enhances cAMP-mediated decidualization of human endometrial stromal cells in an autocrine or paracrine fashion.

Characterization of in vitro Decidualization of Normal Human Endometrial Stromal Cells: Effects of Cell Adhesions

Characterization of in vitro Decidualization of Normal Human Endometrial Stromal Cells: Effects of Cell Adhesions

Autocrine/paracrine regulation of human endometrial stromal remodeling by laminin and type IV collagen

The biological roles of laminin and type IV collagen in human endometrial stromal tissues were investigated by the evaluation of the expression levels in human endometrial tissues using immunohistochemistry. In addition, normal human endometrial stromal cells were cultured in vitro on laminin- or type IV collagen-coated plates and subjected to cytological analyses. Cyclic production of laminin and type IV collagen were detected and the two productions were significantly increased in late proliferative and late secretory endometrial stromal cells. Unstimulated endometrial stromal cells proliferated with specific growth structures that varied depending on the extracellular matrix component coated on the culture plates. The expression levels of integrin subunits on endometrial stromal cells were sufficiently enhanced by 8Br-cAMP treatment to mask any differences in the growth structures induced by the extracellular matrix components. 8Br-cAMP-stimulating stromal cells exhibited significant survival on laminin-coated plates, while 8Br-cAMP-deprived stromal cells, after 8Br-cAMP stimulation, showed significant survival on type IV collagen-coated plates. In conclusion, human endometrial stromal cells produce laminin and type IV collagen, and these productions are possibly regulated by ovarian estrogen and progesterone. Human endometrial stromal cells specifically bind to laminin and type IV collagen via integrins, and regulate endometrial stromal cell structures, viability and differentiation. Thus, laminin and type IV collagen may autoregulate human endometrial stromal remodeling during the menstrual cycle in an autocrine and paracrine fashion.