The effects of estradiol and selective estrogen receptor modulators on gene expression and messenger RNA stability in immortalized sheep endometrial stromal cells and human endometrial adenocarcinoma cells

The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.     

Hormone-dependent changes in A and B forms of progesterone receptor

The influence of different estrogen and/or progesterone treatments on concentrations of A and B forms of progesterone receptor (PR-A and PR-B) in the different cell types of chick oviduct was studied. A semiquantitative immunohistochemical assay for cellular PR concentrations was developed using a computer-assisted image analysis system. The staining intensity of nuclear PR in the basal layer of epithelial cells, glandular, smooth muscle and mesothelial cells was analysed separately using two monoclonal antibodies, PR6 and PR22. The measured concentrations of PR varied between different cell types and from cell to cell. A significant decrease in PR concentration, as noted by a decrease in staining intensity, was observed in all cell types studied 2 or 6 h after a single injection of progesterone with or without simultaneous estrogen administration. The decrease was also verified with immunoblotting and an immunoenzymometric assay (IEMA) for chicken PR. After down-regulation the concentration of PR recovered to the control level within 48 h after progesterone or estrogen administration. Estrogen administration alone was observed to cause changes in the concentration of PR-A only, having little or no effect on PR-B concentration depending on the cell type studied.

These findings indicate that estrogen and progesterone cause cell-specific changes not only to the total concentration of PR but also to the cellular ratio of PR-A and PR-B.     

Ovarian hormone receptors in human mammary stromal cells

Mesenchymal cells of the rodent breast express both estrogen and progesterone receptors. Searches for these molecules in the human breast have yielded conflicting results. Following immunohistochemical staining of samples of normal human breast tissue, the authors detected estrogen receptor alpha protein and progesterone receptor protein in extralobular (non-specialized) fibroblasts and estrogen receptor alpha protein in adipocytes. Tissues from young teenage girls and pregnant women contained the greatest number of receptor positive fibroblasts. These observations confirm prior reports of the presence of ovarian hormone receptors in mammary fibroblasts. The findings also illustrate similarities in the organization of the rodent and human breasts and thereby suggest that regulation of the gland by ovarian hormones involves similar mechanisms in both species.     

不同培养时间对鸡卵泡颗粒细胞孕酮、雌激素分泌水平及FSHR、LHR基因表达的影响

目的：研究培养不同时间鸡卵泡颗粒细胞孕酮和雌激素的分泌水平,促卵泡素受体（FSHR）和促黄体素受体（LHR）的基因表达水平,推断体外培养时间对颗粒细胞激素分泌及相关受体基因表达的影响。方法：通过细胞体外培养的方法,分别于0 h、24 h、48 h、72 h、96 h收集鸡卵泡颗粒细胞上清液,采用ELISA法测定细胞上清液内的孕酮及雌激素分泌水平,并采用荧光定量PCR技术检测颗粒细胞内FSHR和LHR基因表达情况。结果：在培养初期0 h~48 h孕酮和雌激素分泌量显著降低（P < 0.05）,随着培养时间增加到72 h两种激素的分泌量又开始增加,并达到培养初期水平,培养至96 h细胞内孕酮和雌激素分泌量再次降低;颗粒细胞FSHR和LHR mRNA的表达水平则随着培养时间的增加而降低（P < 0.05）。结论：体外培养的卵泡颗粒细胞内孕酮和雌激素的分泌量随体外培养时间的延长呈先降低后升高的趋势,可能与体外培养细胞的生长状态相关,从整体上看随着培养时间的延长,细胞内孕酮和雌激素的分泌量均降低,可能与两种促性腺激素受体FSHR和LHR基因表达量下降相关。     

The Ishikawa cells from birth to the present

More than 20 years have passed since the Ishikawa cell line, a well-differentiated human endometrial adenocarcinoma cell line, was established. Because this cell line bears estrogen and progesterone receptors, the cells have been used in numerous basic research areas such as reproductive biology and molecular science, and has been distributed to more than a hundred institutes. However, even the Ishikawa cells, after long-term culture, tend to transform into undifferentiated cells. In addition, it has been reported that estrogen and progesterone receptors disappeared from the cells that I distributed. I therefore attempted to establish well-differentiated cells from the parent Ishikawa cells and to produce a new and good quality supply of this cell line. I believe that it is very important for the investigator who established a cell line to be responsible for maintaining the quality of the cells. That is why I have not deposited this cell line in any cell bank. I would like to take this opportunity to report the history of Ishikawa cells from establishment to the present.     

Rapid recovery of nuclear estrogen receptor and oxytocin receptor in the ovine uterus following progesterone withdrawal

We previously showed that progesterone rapidly down regulates nuclear estrogen receptor (Re) in the estrogen-primed rodent uterus. We have now extended these studies to test the response of the Re system in sheep uterus to progesterone withdrawal. Since the estrogen-Re complex is believed to regulate hormone-dependent gene expression, it was of interest to determine whether withdrawal of progesterone under constant estrogen stimulation would lead to the recovery of nuclear Re levels and estrogen action, i.e. oxytocin receptor (ROT) synthesis. Ovariectomized ewes were primed with estradiol-17 beta and serum steroid levels were maintained by constant infusion of estradiol (0.5 microgram/h) and progesterone (500 micrograms/h) for 5 days. The animals were anesthetized with fluothane/O2, and uterine samples were excised 1 h before and 3, 6 and 12 h after progesterone withdrawal. Estradiol infusion was continued during the experiment in order to maintain estrogen levels at a steady state (14 pg/ml plasma). Re, ROT and progesterone receptor (Rp) were measured in endometrium and myometrium using standard 3H-hormone binding assays. Following progesterone withdrawal, the nuclear Re concentration increased in both uterine compartments, and the nuclear Re level was correlated significantly with the ROT concentration in the membrane fraction of both uterine tissues (endometrium, r = 0.79; myometrium, r = 0.86). Although cytosol Re rose between 6 and 12 h in the endometrium, cytosol Re levels remained unchanged in myometrium. Cytosol Rp appeared to increase in endometrium but not in myometrium. Uterine tissue sampled from a control animal before stopping the progesterone infusion revealed that the observed changes in receptor concentration following progesterone withdrawal were not due to regional differences in receptor levels. These results demonstrate that the recovery of nuclear Re in the ovine endometrium and myometrium following progesterone withdrawal represents a selective effect on Re retention in the nucleus rather than on cytosol Re availability or Re activation which was controlled by constant estrogen infusion. Thus, these results are consistent with the hypothesis that progesterone induces an Re regulatory factor which acts to down regulate nuclear Re, and that the activity of this factor diminishes rapidly after progesterone withdrawal.     

Tamoxifen decreases the estradiol induced progesterone receptors by interfering with nuclear estrogen receptor accumulation

The retention time of the estrogen receptor in the nucleus of target cells after antiestrogen treatment has been shown to be longer than after estradiol. This paper describes the accumulation of nuclear estrogen receptors and the obtention of estrogenic responses (i.e. synthesis of cytosolic progesterone receptors and DNA) in the rat uterus after tamoxifen treatment in the presence or absence of estradiol. One-week ovariectomized adult rats were implanted with a silicone elastomer capsule containing corn oil or 25 micrograms estradiol/capsule (0 h). 48 h after implantation rats were injected with corn oil or 2 mg tamoxifen/kg and decapitated at 72, 96 or 120 h after implantation. In parallel experiments the implants were removed just before the injections of tamoxifen or oil. Tamoxifen injected into rats implanted with oil increased both the occupied nuclear receptors and the progesterone receptors at 96 h. In rats implanted with estradiol, tamoxifen did not increase the occupied nuclear receptors and decreased the levels of progesterone receptor and DNA at 96 h. In rats whose estradiol implants were removed at 48 h tamoxifen did not change the level of occupied nuclear receptors at 72 h but it increased them abruptly at 96 and 120 h. In these rats progesterone receptors decreased at 72 h but they increased at 96 and 120 h, and DNA decreased at 120 h to a lower level than before implantation. The results suggest that when estradiol is acting, tamoxifen is not able to increase the level of occupied estrogen receptor and it acts as an antiestrogen by decreasing the high level of progesterone receptors previously induced by estradiol. When estradiol is not acting tamoxifen behaves as a partial estrogen agonist by inducing progesterone receptors. However, the antiestrogenic action of tamoxifen on the rat uterus DNA does not seem to be affected by estradiol.     

Progesterone receptor replenishment in T47D human breast cancer cells. Roles of protein synthesis and hormone metabolism

T47D are unusual human breast cancer cells that do not require estrogen to synthesize high levels of progesterone receptors. These cells can, therefore, be used to study the mechanisms by which progesterone, freed of estrogen interference, controls the synthesis of its receptors. In a recent paper we described progesterone receptor translocation and a subsequent very rapid nuclear processing step that results in an apparent loss of 60 to 80% of cellular progesterone receptors, 30 to 60 min after progesterone treatment. This paper deals with the replenishment of cellular receptors following processing. If progesterone is removed from cells after 60 min of treatment, cytoplasmic progesterone receptors replenish in 16 to 20 h. However, replenishment occurs even during chronic progesterone treatment; this is an artifact created by the extremely rapid (t1/2 approximately 2 h) metabolism of progesterone in media exposed to cells. If progesterone metabolism is blocked, then replenishment is not seen, probably because the hormone continuously retranslocates the newly replenished sites. There is an early protein synthesis-dependent step; cycloheximide in the first 4 h inhibits replenishment 24 h later, but if cycloheximide is slightly delayed (beyond 4 h), replenishment proceeds normally. In contrast to progesterone, the synthetic progestin R5020 completely suppresses progesterone receptor replenishment even 96 h after its removal from the medium. This compound can bind covalently to receptors and may be very difficult to remove from cells. Clearly, progestin treatment, and by analogy, circulating progesterone, will have profound effects on cytoplasmic and nuclear progesterone receptor levels when these are measured in biopsied human tumors as an adjunct to endocrine therapy.     

Regulation of SULT1E1 expression in Ishikawa adenocarcinoma cells by tibolone

Tibolone is used therapeutically as a hormone replacement agent and has beneficial effects on osteoporosis and hot flushes as well as libido in post-menopausal women without stimulatory effects in the breast and endometrium. The lack of effect in the endometrium is due in part to the tissue specific sulfation of tibolone and its active metabolites in endometrial tissues. Tibolone is metabolized into 3alpha-OH and 3beta-OH tibolone as well as the Delta4-isomer. Tibolone and the Delta4-isomer bind and activate progesterone and androgen receptors whereas 3alpha-OH and 3beta-OH tibolone activate the estrogen receptors. Human endometrium and Ishikawa endometrial adenocarcinoma cells express SULT1E1 that efficiently sulfates both 3-OH tibolone metabolites and has trace activity with tibolone but no activity with the Delta4-isomer. Treatment of Ishikawa cells with all four tibolone compounds resulted in the induction of SULT1E1 activity similar to the induction by progesterone. The induction of SULT1E1 was inhibited by RU486 indicating a role for the progesterone receptor. Sulfation of the tibolone compounds by Ishikawa cells and Ishikawa cells expressing physiological levels of SULT1E1 activity resulted in the sulfation of tibolone and the 3-OH metabolites but not Delta4-tibolone. These results indicate that the lack of endometrial stimulation involves induction of SULT1E1 and the selective sulfation and inactivation of the estrogenic 3-OH tibolones and interconversion of the tibolone metabolites to generate the progestagenic non-sulfated Delta4-isomer.     

Immunohistochemical detection of estrogen receptors in the canine uterus and their relation to sex steroid hormone levels

Cyclic changes in estrogen receptor expression in the uterine tissue of 60 female dogs were evaluated, using an immunohistochemical technique on formalin-fixed paraffin-embedded sections. The expression of estrogen receptors in the uterine horns, body and cervix was quantified by means of an immunohistochemical score. A negative correlation was found between staining scores in the uterine horns and serum progesterone levels. Generally, staining scores in the uterine horns were highest during proestrus, declined during estrus and were lowest during early metestrus. During anestrus high staining scores for estrogen receptors were observed, indicating sensitivity for estrogens in a sexual quiescence stage. Compared with the uterine horns, high staining scores were found in the uterine body and cervix during estrus and metestrus. No positive staining for estrogen receptors was detected in 1 pregnant uterus. Fluctuations in estrogen receptors were more pronounced in endometrial stroma cells than in epithelial cells of the uterine horns. The importance of stromal cells in the sexual cyclicity of the canine uterus should not be underestimated when studying uterine endocrinology and pathology.     

Quantitative image analysis of estrogen and progesterone receptors as a prognostic tool for selecting breast cancer patients for therapy

OBJECTIVE: To assess estrogen and progesterone receptor presence in human breast tumors using immunocytochemical analysis. STUDY DESIGN: For both estrogen (ER) and progesterone (PR) receptor assay, percent of stained cells and intensity of staining were estimated on a series of 251 consecutive breast cancer cases from the M. Ascoli Cancer Hospital Center in Palermo using the CAS 200 image analysis system. RESULTS: Cytochemical assay revealed a differential distribution of both ER and PR, by menopausal status of the patients; premenopause (PreM) was mostly ER negative (63%), and postmenopause (PostM) > 10 years was mostly ER and PR positive (64%). The percent of cells stained for ER was significantly different between PreM and PostM patients when they were considered as a whole. By contrast, no difference emerged for PR staining among menopausal groups. Overall, patients whose tumors were PR positive showed a significantly (P < .03) longer interval free of relapse. CONCLUSION: The present results suggest that PRs behave as better indicators than ERs of early relapse in breast cancer patients. Further studies, with longer follow-up, are needed, however, to validate this concept.     

Hormonal regulation of estrogen and progestin receptors in decidual cells

Total estrogen receptor (Re) and total progestin receptor (Rp) were measured in the cytosol and nuclear fractions from hamster deciduomal tissue and decidual cell cultures. Correlation of serum steroid (estradiol and progesterone) and deciduomal receptor profiles revealed a significant loss of Re during the first four days of decidualization that was not attributable to changes in serum steroid levels. A decidual cell-tissue culture system was used to study the receptor's recovery response to progesterone withdrawal. Decidual cells were plated and grown in Ham's F12/Dulbecco's modified Eagle's medium with 5% horse serum supplemented with insulin, transferrin, selenium and progesterone (10 ng/ml). Within 48 h of culture large, multinucleate decidual cells were observed by phase microscopy. At 72 h of culture in medium containing progesterone, only Rp was detectable in decidual cells. Re was not detectable (less than 200 fmol/mg DNA) in either cytosol or nuclei from cells maintained in the presence of progesterone. However, when progesterone was deleted from the medium, cytosol Re recovered progressively from 8 h to 16 h of culture. Progesterone withdrawal also caused parallel increases in cytosol and nuclear Rp, and estradiol treatment (2 ng/ml) in combination with progesterone withdrawal further enhanced Rp levels in decidual cell cultures. These results with cultured decidual cells demonstrate that progesterone down-regulates Re and Rp, Re recovers rapidly upon progesterone withdrawal, and the Re system is competent to respond to estrogen action in terms of Rp induction. We used the density-shift method to determine that progestin increases the turnover of nuclear Re in hamster decidual cells within 3 h. Hamster decidual cells were isolated from the endometrium and cultured in progesterone-free medium containing normal amino acids (1H, 12C, 14N) for 2 days. Confluent monolayers of cells were exposed to 1 nM estradiol (E2) for 1 h to maximize the amount of occupied Re in the nuclear fraction. Then, at time 0, cells were transferred to medium supplemented with dense (2H, 13C, 15N) amino acids and either 1 nM E2 or E2 plus 100 nM progesterone. After Re was labeled with dense amino acids for 1, 3, 6 and 9 h, nuclear Re was extracted with 10 mM pyridoxal -5' phosphate and labeled with 125I-iodoestradiol (5 nM). Two radioactive peaks representing preexisting and newly synthesized Re were separated by sucrose density-gradient centrifugation. The halflife of nuclear Re in decidual cells was 3.7 h when cells were treated with E2 alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

人早孕子宫蜕膜催乳素分泌的调节

子宫内膜蜕膜化对胚泡植入与妊娠维持是非常重要的。为探讨蜕膜化维持的调节机制 ,本文研究了妊娠早期人子宫蜕膜细胞催乳素 (PRL)分泌的调节。结果表明 :(1)孕酮显著地刺激PRL的分泌。 (2 )雌激素的作用与其浓度有关 ,生理浓度的雌激素对PRL分泌无明显影响 ,而高水平的雌激素抑制孕酮的刺激作用。合适的雌孕激素比例对蜕膜化的维持是必要的。 (3)RU486明显地抑制PRL的分泌 ,故认为孕酮的作用至少是部分通过受体介导的机制。 (4 )高浓度的cAMP (≥ 10 -5mol/L)显著增加PRL的分泌 ,cAMP信号系统可能在蜕膜化反应中发挥重要作用。     

Progesterone abbreviates the nuclear retention of estrogen receptor in the rat oviduct and counteracts estrogen action on egg transport

Progesterone has synergistic or antagonistic effects on several estrogenic actions. The effects of progesterone on estrogen-induced accelerated ovum transport and on the dynamics of estrogen receptors in the rat oviduct were examined. The involvement of the progesterone receptors in these phenomena was assessed. On Day 1 of pregnancy, rats were treated with estradiol, estradiol plus progesterone, or either one plus the progesterone receptor-blocking agent RU486. Control animals received the oil vehicle alone. The number of eggs remaining in the oviduct was assessed 24 h after treatment. Cytoplasmic and nuclear estrogen receptor levels in the oviduct, as well as plasma concentrations of estradiol and progesterone, were measured at various intervals--up to 11 h and 24 h after treatment, respectively. Accelerated oviductal egg transport induced by estrogen was blocked by the concomitant administration of progesterone. This effect of progesterone was not associated with changes in estrogen circulating levels and was preceded by a reduction in the total amount of estrogen receptors and by a shortened retention of estrogen receptors in the nucleus. The effects of progesterone on egg transport and on the levels of estrogen receptors were reversed by blocking the progesterone receptor with RU486, suggesting that both effects were receptor-mediated. These findings demonstrate that progesterone antagonizes the effect of estrogen on oviductal egg transport in the rat, and suggest that this antagonism is mediated by a reduction both in the amount of estrogen receptors and in their retention time in the nucleus.     

17 beta-estradiol activates PI3K/Akt signaling pathway by estrogen receptor (ER)-dependent and ER-independent mechanisms in endometrial cancer cells

Cellular response to estrogen is mediated both by estrogen receptor (ER) binding to estrogen response element (ERE) and by non-nuclear actions like activation of signal transducing pathways. The main aims are to study if PI3K/Akt signaling pathway can be activated by 17beta-estradiol (E2) via non-nuclear action and to investigate the relationship of the action of E2 and ER in endometrial cancer cells expressing with different status of ER. The levels of phosphorylated Akt (Ser473) (P-Akt) and total Akt were examined by western blot and Akt kinase activity was measured in cells after stimulation with 1 microM E2 at different time points. Inhibitory role of LY294002 on activation of Akt induced by E2 and its estrogen antagonist, ICI182780 were also tested. P-Akt/Akt was used as a measure of activation of Akt. We found that maximum P-Akt/Akt and Akt kinase activity took place at 30 min in Ishikawa cells and 15 min in HEC-1A cells and the activation persisted for at least 2 h after stimulation with 1 microM E2. The activation of Akt elicited gradually with increasing doses of E2. PI3K inhibitor, LY294002, stopped the activating Akt in a dose-dependent manner and 50 microM LY294002 completely blocked the activation of Akt induced by E2. ICI182780 could block the activation of PI3K/Akt in ER-positive Ishikawa cells but not in HEC-1A cells with poor-expressed ER. This study demonstrated that E2 is able to promptly activate PI3K/Akt signal pathway in Ishikawa cells in an ER-dependent manner and ER-independent in HEC-1A cells. Blockage of PI3K/Akt cascade may become a potential and effective way to control endometrial carcinoma, especially in ER-negative cancers, which show no response to endocrinal therapy.     

In vitro canine oocyte nuclear maturation in homologous oviductal cell co-culture with hormone-supplemented media

The aim of the present research was to verify the influence of oviductal cell co-culture previously supplemented with steroids (estrogen, progesterone, or both) on IVM rates for oocytes from anestrous bitches that were cultured in vitro for 48, 72 and 96 h. Oocytes harvested from anestrous bitches were selected and allocated into four groups: Group 1 (co-culture in oviductal epithelial cells without hormonal supplementation-control); Group 2 (estrogen supplementation); Group 3 (progesterone supplementation); Group 4 (estrogen+progesterone supplementation). The oviductal epithelial cell culture was established 72 h prior to oocyte co-culture. After periods of 48, 72 and 96 h, the degree of oocyte nuclear maturation was assessed. Co-culture in oviductal epithelial cells with estrogen was not as beneficial for canine IVM as supplementation with progesterone and estrogen, or progesterone supplementation alone. Therefore, it was feasible to use co-culture with oviductal epithelial cells obtained from anestrous bitches for IVM (monolayer culture with oviduct cells previously supplemented with progesterone). Final stages of oocyte maturation were achieved at 72 and 96 h of culture; therefore, the duration of maturation for oocytes obtained from bitches in different stages of the estrous cycle should be taken into account.     

Stimulatory effect of estrogen on the growth of endometrial cancer cells is regulated by cell-cycle regulators

Estrogen is known as a major risk factor in tumorigenesis of the endometrium. The aim of this study is to establish stable estrogen-responsive endometrial cancer cell lines and to investigate the mechanism of estrogen action, focusing on cell-cycle regulation. Human wild-type estrogen receptor cDNA was transfected into endometrial cancer cells (Ishikawa) and estrogen-responsive cell lines were cloned. Their estrogen responsiveness was evaluated by the effect of estrogen on cellular growth and progesterone receptor expression. It was quantitatively estimated by immunocytochemistry or immunoblotting how the expression of cell-cycle regulators such as cyclin D1, cyclin E, Cyclin A, p53, p21 and p27 was regulated by estrogen. A cell line stably responsive to estrogen was established, and cells proliferated and the glandular structure was formed by estrogen stimulation. Cyclin D1 expression increased at 6–24 h and cyclin A gradually increased until 48 h of estrogen treatment compared with untreated cells. On the other hand, p53 and p21 expressions decreased at 6–24 h, and p27 gradually decreased until 24 h by estrogen. Our results show that the stimulatory effect of estrogen on cell proliferation may be regulated by the up-regulation of cyclin D1 and cyclin A, and down-regulation of p53, p21 and p27. This cell line is useful to clarify the molecular mechanism of estrogen action on endometrial cancer.