A novel selective progesterone receptor modulator asoprisnil (J867) down-regulates the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured uterine leiomyoma cells

BACKGROUND: This study was conducted to evaluate the effects of a novel selective progesterone receptor modulator (SPRM) asoprisnil on the expression of growth factors and their receptors and on growth factor-induced proliferation of cultured uterine leiomyoma and matching myometrial cells. METHODS: The expression of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and transforming growth factor (TGFbeta3) was assessed by immunocytochemistry and semi-quantitative RT-PCR. The expression of phosphorylated EGF receptor (p-EGFR), IGF-I receptor alpha subunit (IGF-IRalpha) and phosphorylated TGFbeta receptor type II (p-TGFbeta RII) was assessed by Western blot analysis. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. RESULTS: Treatment with 10(-7) M asoprisnil decreased EGF, IGF-I and TGFbeta3 mRNA and protein expression as well as p-EGFR, IGF-IRalpha and p-TGFbeta RII protein expression in leiomyoma cells cultured for 72 h. EGF (100 ng/ml), IGF-I (100 ng/ml) and TGFbeta3 (10 ng/ml) increased the number of viable leiomyoma cells cultured for 72 h, whereas the concomitant treatment with 10(-7) M asoprisnil antagonized the growth factor-induced increase in leiomyoma cell proliferation. In cultured myometrial cells, however, asoprisnil affected neither the growth factor and their receptor expression nor the cell proliferation. CONCLUSION: Asoprisnil inhibits the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured leiomyoma cells without affecting their expressions in myometrial cells.     

Progesterone receptor modulator CDB-2914 down-regulates vascular endothelial growth factor, adrenomedullin and their receptors and modulates progesterone receptor content in cultured human uterine leiomyoma cells

BACKGROUND: This study was conducted to evaluate the effects of graded concentrations (10(-8), 10(-7) and 10(-6) M) of progesterone receptor (PR) modulator CDB-2914 on the protein contents of PR, of vascular endothelial growth factor (VEGF), adrenomedullin (ADM) and their receptors in cultured human uterine leiomyoma and matching myometrial cells. METHODS: PR-A, PR-B, VEGF-A, VEGF-B, VEGF receptor (VEGFR)-1, VEGFR-2, ADM and ADM receptor (ADMR) contents were assessed by Western blot analysis. RESULTS: Treatment with 100 ng/ml progesterone increased VEGF-A, VEGF-B and ADM contents in cultured leiomyoma cells and normal myometrial cells. The concomitant treatment with 10(-6) M CDB-2914 significantly decreased the progesterone-induced VEGF-A, VEGF-B and ADM contents in cultured leiomyoma cells but not in normal myometrial cells. CDB-2914 treatment alone decreased VEGFR-1, VEGFR-2 and ADMR contents in cultured leiomyoma cells but not in normal myometrial cells. CDB-2914 treatment increased PR-A and decreased PR-B contents in cultured leiomyoma cells in a dose-dependent manner compared with untreated cultures, whereas no significant changes in PR isoform contents were observed in normal myometrial cells. CONCLUSIONS: These results suggest that CDB-2914 down-regulates VEGF, ADM and their receptor contents and modulates PR isoform contents in cultured leiomyoma cells in a cell-type-specific manner.     

Progesterone receptor modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells

Effects of progesterone receptor modulator CDB-2914 on the expression of the extracellular matrix (ECM) components were examined in cultured human uterine leiomyoma and myometrial cells. ECM metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagen levels were assessed by Western blot analysis, MMP activity assay and real-time RT-PCR. RNA interference (RNAi) of EMMPRIN was performed using small interfering mRNA. In cultured leiomyoma cells, CDB-2914 treatment at concentrations greater than or equal to 10(-8) M significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium. TIMP-1 and TIMP-2 were significantly decreased at mRNA and protein levels by CDB-2914 treatment at concentrations > or =10(-7) M in these cells. CDB-2914 treatment decreased types I and III collagen protein contents. However, CDB-2914 treatment did not affect the ECM component expression in cultured myometrial cells. RNAi of EMMPRIN abrogated CDB-2914-mediated both induction of MMPs and reduction of TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914 modulates the expression of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells without comparable effects on myometrial cells.     

Comparative effects of heparin-binding epidermal growth factor-like growth factor on the growth of cultured human uterine leiomyoma cells and myometrial cells

BACKGROUND: The objective of this study was to investigate the comparative effects of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on the growth of cultured human leiomyoma cells and myometrial cells. METHODS: Isolated cells were subcultured in Phenol Red-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for an additional 24 and 48 h in the presence or absence of graded concentrations of HB-EGF (0.1, 1, 10 and 100 ng/ml). These cells were used for immunocytochemical analysis for Ki67, western blot analysis for proliferating cell nuclear antigen (PCNA) and human EGF receptor (HER1), and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay. RESULTS: Treatment with HB-EGF at concentrations >1 ng/ml significantly increased the Ki67-positive rate of cultured leiomyoma cells and myometrial cells. Treatment with HB-EGF also resulted in a dose-dependent increase in PCNA expression in both cells compared with untreated control cultures. A significant increase in PCNA expression in cultured myometrial cells was noted following treatment with HB-EGF at concentrations >1 ng/ml, whereas an increase in PCNA expression in cultured leiomyoma cells was noted following treatment with HB-EGF at concentrations >10 ng/ml. HER1 expression was significantly higher in untreated myometrial cells than in untreated leiomyoma cells. A significant increase in HER1 expression in myometrial cells was observed when treated with HB-EGF at concentrations >10 ng/ml, whereas a significant increase in HER1 expression in leiomyoma cells was noted only by the treatment with HB-EGF at concentrations >100 ng/ml. Treatment with HB-EGF decreased the TUNEL-positive rate of those cells with no significant differences between the two cell types. CONCLUSIONS: The results obtained suggest that HB-EGF plays a role in stimulating the proliferation of leiomyoma cells and myometrial cells and in inhibiting apoptosis of those cells through augmentation of HER1 expression. Since the proliferative potential of myometrial cells responded better to HB-EGF than that of leiomyoma cells, HB-EGF may play a more vital role in myometrial growth than leiomyoma growth.     

Selective progesterone receptor modulator asoprisnil down-regulates collagen synthesis in cultured human uterine leiomyoma cells through up-regulating extracellular matrix metalloproteinase inducer

BACKGROUND: A recent clinical trial demonstrated that selective progesterone receptor modulator asoprisnil is effective in reducing uterine leiomyoma volume. We investigated the effects of asoprisnil in vitro on the expression of the extracellular matrix (ECM)-remodeling enzymes and collagens in cultured leiomyoma and matching normal myometrial cells. METHODS: The expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagens were assessed by western blot analysis. RESULTS: Untreated cultured leiomyoma cells had significantly lower EMMPRIN (P < 0.05), MMP-1 (P < 0.05) and membrane type 1-MMP (MT1-MMP) (P < 0.01) protein contents, but significantly higher TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.05) and type III (P < 0.01) collagen protein contents compared with untreated cultured myometrial cells. Treatment with asoprisnil at concentrations > or =10(-7) M for 48 h significantly (P < 0.05) increased EMMPRIN, MMP-1 and MT1-MMP protein contents, and decreased TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.01) and type III (P < 0.05 at 10(-7) M; P < 0.01 at 10(-6) M) collagen protein contents in cultured leiomyoma cells compared with control cultures. However, asoprisnil treatment did not affect the protein contents of ECM-remodeling enzymes and collagens in cultured myometrial cells. CONCLUSIONS: These results suggest that asoprisnil may reduce collagen deposit in the ECM of cultured leiomyoma cells through decreasing collagen synthesis and enhancing the expression of EMMPRIN, MMPs and TIMPs without comparable effects on cultured myometrial cells.     

The effects of progesterone on apoptosis in the human trophoblast-derived HTR-8/SV neo cells

Progesterone (P4) is frequently used in the treatment of threatened abortion, prevention of recurrent miscarriage and threatened preterm labor. However, little is known about the molecular mechanism of P4 in the regulation of extravillous trophoblasts' (EVTs) function. This study was designed to examine the presence of progesterone receptor (PR) in the human trophoblast-derived HTR-8/SV neo cell line, which is a possible model of EVTs, and the effects of P4 on apoptosis in those cells. The HTR-8/SV neo cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 microg/ml streptomycin. When the cell the population reached 50% confluency, the cells were stepped down to serum-free conditions in the presence or absence of graded concentrations of P4 (1, 10 and 100 ng/ml) for 48 h. The cultured cells were used for RT-PCR, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay, immunocytochemistry and western blot analyses. Immunocytochemistry and western blot analyses revealed that PR was evident in HTR-8/SV neo cells. Compared with untreated cultures, treatment with P4 (10 and 100 ng/ml) resulted in significant decreases in the TUNEL-positive rate, Fas, Fas ligand (Fas-L), caspase-8, caspase-3 and poly (ADP-ribose) polymerase (PARP) expression in HTR-8/SV neo cells, and a significant increase in Bcl-2 expression in those cells. Consistently, Fas mRNA expression in those cells was significantly inhibited by the treatment with 10 ng/ml P4 compared with untreated cultures. This study suggests that PR exists in HTR-8/SV neo cells and that P4 inhibits apoptosis by down-regulating Fas, Fas-L, caspase-8, caspase-3 and PARP expression as well as up-regulating Bcl-2 expression in HTR-8/SV neo cells.     

Progesterone production by human granulosa cells cultured in serum free medium: effects of gonadotrophins and insulin-like growth factor I (IGF-I).

There is evidence that insulin-like growth factor I (IGF-I) is a potent regulator of oestradiol synthesis by human granulosa and luteal cells; however, the question of whether IGF-I regulates progesterone synthesis by these cell types has yet to be answered. As a first step towards this goal, we have compared the effects of IGF-I, follicle stimulating hormone (FSH), and human chorionic gonadotrophin (HCG) on progesterone production by human granulosa cells obtained from individual dominant and cohort follicles, and granulosa luteal cells from preovulatory follicles of patients undergoing in-vitro fertilization (IVF). Granulosa cells from normal, unstimulated follicles cultured in serum-free medium as controls (no additions) produced some progesterone spontaneously. In all cases, FSH stimulated basal progesterone levels (10-fold average increase) and the effect was dose-dependent (ED50 of FSH = 9.1 +/- 3.9 ng/ml). Similar effects were observed when granulosa cells from large follicles were incubated with HCG (ED50 of HCG = 6.9 +/- 2.8 ng/ml). By comparison, the effects of IGF-I on progesterone production were not marked, being absent in 80% of the follicles tested. However, granulosa cells from healthy follicles co-incubated with IGF-I and FSH or HCG produced more progesterone compared with cells treated with the gonadotrophins alone; this effect of IGF-I was dose dependent (ED50 of IGF-I = 10 ng/ml). When the effect of each agonist was tested on IVF granulosa luteal cells, HCG but not FSH or IGF-I stimulated basal progesterone levels but the HCG effect required a two-day lag phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

ORIGINAL ARTICLE: Effect of Progesterone on HLA‐E Gene Expression in JEG‐3 Choriocarcinoma Cell Line

Problem Among class Ib human leukocyte antigen (HLA) molecules, HLA‐E is known to be a major ligand of CD94/NKG2 receptor on natural killer (NK) cells, and to play a pivotal role in recognition of extravillous trophoblasts (EVTs) by maternal immune cells. However, it is scarcely known how HLA‐E expression is regulated in EVTs. Method of study In this study, we investigated whether progesterone, an essential hormone in maintaining pregnancy, regulated HLA‐E expression in EVT‐like cell line, JEG‐3. HLA‐E mRNA amount in cultured JEG‐3 cells was assessed by real‐time PCR and cell‐surface HLA‐E protein was analyzed by flowcytometry. Results Real‐time PCR showed 3.5‐fold increase 1 hour after the addition of 1000 ng/ml progesterone. This response was dimished by the addition of RU486, an antagonist for progesterone receptor. Flowcytometry indicated that 1000 ng/ml progesterone slightly enhanced HLA‐E expression on the surface of JEG‐3. Conclusion These results suggest that progesterone up‐regulates HLA‐E expression in JEG‐3 cells through the pathway mediated by progesterone receptor. Our findings might give a new insight into immunomodulatory function of progesterone at fetomaternal interface.     

Estrogen and progesterone receptor expression in macrophages and regulation of hepatocyte growth factor by ovarian steroids in women with endometriosis

BACKGROUND: Information regarding macrophage-mediated regulation of hepatocyte growth factor (HGF) by ovarian steroid hormones in women with endometriosis is limited. Therefore, we investigated the regulation of HGF by steroid hormones in isolated macrophages and stromal cells derived from women with or without endometriosis. METHODS: We isolated CD68 immunoreactive adherent macrophages in vitro from 46 women with endometriosis and 30 women without endometriosis. Estrogen receptor (ER) and progesterone receptor (PR) expression in macrophages was demonstrated by immunohistochemistry and RT-PCR. Production of HGF in the culture media of basal and ovarian steroid-stimulated macrophages was examined by enzyme-linked immunosorbent assay. Expression of mRNA for HGF and its receptor, c-Met in macrophages and stromal cells in response to ovarian steroid was investigated by RT-PCR. The single and combined effect of HGF and estrogen on the growth of macrophages and stromal cells was analysed by bromodeoxyuridine (BrdU) incorporation. RESULTS: ER and PR were expressed in isolated macrophages and intact tissue at the protein and mRNA levels. Macrophages derived from women with endometriosis produced significantly higher concentration of HGF (352.2 +/- 4.9 pg/ml) in conditioned media after treatment with estradiol (10(-8) mol/l) than that of basal macrophages (221.5 +/- 32.8 pg/ml, P<0.05) or women without endometriosis (170.6 +/- 2.6 pg/ml, P<0.05). These effects were less evident after treatment with progesterone. Treatment with tamoxifen (10(-6) mol/l) reversed the production of HGF and other macromolecules. Secretion of HGF in response to ovarian steroids was further enhanced after activation with lipopolysaccharide. The mRNA expressions of HGF and its receptor, c-Met, were also detected in macrophages and stroma in response to estrogen, suggesting an autocrine regulation. HGF mRNA expression was higher in cells of women with endometriosis than non-endometriosis women. Bromodeoxyuridine incorporation indicated that exogenous stimulation with HGF and estrogen, either alone or in combination, significantly increased the cell proliferation of both endometrial stroma and macrophages compared to that of non-endometriosis or non-treated cells. CONCLUSION: These results suggest that besides other inflammatory mediators, ovarian steroids also participate in the production of HGF by peritoneal macrophages which may be involved in the growth of endometriosis either alone or in combination with estrogen.     

Increased expression of latent TGF-beta binding protein-1 and fibrillin-1 in human uterine leiomyomata

We compared latent TGF-ss binding protein-1 (LTBP-1) and fibrillin-1 (FBN-1) expression in leiomyomata and myometrium, correlated with leiomyomata size. We studied in vivo and in vitro effects of ovarian steroids using matched leiomyomata and myometrium samples from both phases of the menstrual cycle. Leiomyomata were divided into small (or=6 cm) groups. We validated LTBP-1 and FBN-1 expression using QPCR, western blot and immunohistochemistry. LTBP-1 and FBN-1 mRNA and protein expressions were higher in the medium-sized group compared with myometrium in the proliferative phase (P = 0.01; P = 0.01). FBN-1 mRNA expression was higher in the secretory phase (P = 0.01). LTBP-1 mRNA and protein expression was higher in the medium group compared with the small and large groups in the proliferative phase (P = 0.04; P = 0.04). No differences between groups were seen in FBN-1 expression in either phase. 17Beta-estradiol (E2) increased mRNA and protein expression of LTBP-1 and FBN-1 in cultured leiomyoma smooth muscle cells (LSMC) (P < 0.05). No change in FBN-1 and LTBP-1 expression was observed when cells were treated with E2 plus progesterone. Estrogen may be involved in LTBP-1 and FBN-1 expression in leiomyomata. Extracellular matrix metabolism may be different in medium-sized leiomyoma.     

A novel organotypic culture model for normal human endometrium: regulation of epithelial cell proliferation by estradiol and medroxyprogesterone acetate

BACKGROUND: A novel organotypic culture system was established for modelling the hormonal responses of the normal human endometrium in vitro. METHODS: Endometrial epithelial cells were cultured as glandular organoids within reconstituted extracellular matrix (Matrigel) in tissue culture inserts and stromal cells on plastic below the epithelial compartment. The effects of estradiol (E2) and E2 together with medroxyprogesterone acetate (MPA) on cell proliferation and the expression of estrogen receptor alpha (ERalpha) and progesterone receptor (PR) were studied in 10 epithelial-stromal co-cultures and in three parallel monocultures of epithelial organoids. RESULTS: In co-cultures, E2 was shown to increase the percentage of Ki67-positive cells by approximately 2-fold relative to untreated controls. In the presence of MPA, a significant decrease in cell proliferation was detected. Similar results were obtained when the corresponding percentages of Ki67-positive organoids were calculated instead of individual cells. In the absence of stromal fibroblasts, Ki67 epithelial labelling remained below the control value after both hormonal treatments. Epithelial organoids retained their capacity to express estrogen and progesterone receptors in culture. E2 was shown to markedly increase and MPA to down-regulate the expression of PR. The expression of ERalpha was only slightly affected by either hormonal treatment. CONCLUSIONS: The present organotypic model provides a novel in vitro system in which to study the effects of steroids in the normal human endometrium both in terms of cell proliferation and gene expression. The culture system holds promise as a useful method to screen novel steroid compounds and may help to circumvent problems related to the use of animal models.     

Ovarian steroids and the human breast: regulation of stem cells and cell proliferation

Ovarian steroidal control of mammary gland proliferation and differentiation is not well defined in the human. We therefore developed the athymic nude mouse model in which intact normal human breast tissue is xenografted subcutaneously and treated with human physiological serum levels of oestrogen (E) and/or progesterone (P). We showed that: (i) E, and not P, is the major steroid hormone inducing proliferation of epithelial cells in the adult non-pregnant, non-lactating breast; (ii) E induces progesterone receptor (PR) expression; and (iii) PR expression is maximally induced at low E concentrations while a higher amount of E was required to induce proliferation. Using double label immuno-fluorescence, we demonstrated that cells expressing the oestrogen receptor- (ER) invariably contained the PR but that steroid receptor expression and cell proliferation (Ki67 antigen) were dissociated. Recently, we have demonstrated that some ER/PR-positive epithelial cells are quiescent breast stem cells suggesting that they act as “steroid hormone sensors” that secrete paracrine factors to regulate the proliferative activity of adjacent ER/PR-negative epithelial cells. The dissociation between steroid receptor expression and cell proliferation in normal epithelium was lost at an early stage in ER/PR-positive breast tumour formation perhaps indicating that they arise from deregulation of the normally quiescent breast stem cells.     

内异症子宫内膜ER、PR基因表达的研究

目的/:v探讨子宫内膜异位症(内异症)子宫内膜雌激素受体(ER)、孕激素受体(PR)基因表达在内异症发病中的作用。方法:利用大鼠内异症动物模型,采用逆转录聚合酶链反应(RT-PCR)技术,检测子宫内膜ER和PRmRNAs的表达情况。结果:内异症模型组大鼠异位内膜ER、PRmRNAs的表达显著低于在位内膜及对照组正常子宫内膜(P＜0.01);而模型组在位内膜ER、PRmRNAs的表达与正常对照组比较差异无显著(P＞0.05)。内异症模型组异位内膜ER/PRmRNA比值大于在位内膜及正常子宫内膜ER/PRmRNA比值(P＜0.01)。结论:内异症大鼠异位内膜ERmRNA表达的相对增高在内异症的发生与发展中起着一定的作用。