Sexual dimorphism of steroid hormone receptor messenger ribonucleic acid expression and hormonal regulation in rat vascular tissue.

Evidence has accumulated suggesting that steroid hormones have a direct effect on the vascular system. Of special interest is the protective effect of estrogens against cardiovascular diseases. One of the aims of the present study was to investigate the messenger RNA (mRNA) expression of the five classic steroid hormone receptors in the great vessels of the rat (aorta, vena cava, and vena portae) to provide a basis to analyze direct steroid effects on vascular tissue. By applying reverse transcription-PCR we compared the expressions of the steroid hormone receptor mRNAs in the respective vessels of male and female rats. Sex differences in the mRNA levels of the mineralocorticoid (MR), estrogen (ER), and progesterone (PR) receptors were found in venous vessels, but not in the aorta. Focussing on the vena cava in the female rat, we investigated whether the ER is active in the vasculature by analyzing regulation of the PR gene. This gene is known to be regulated by estrogens in classic target organs. PR mRNA expression in venous vessels of female rats decreased after ovariectomy. This effect was reversed by chronic sc treatment with estradiol (E2; 1 microgram/animal day). Progester-one (10 mg/animal day, sc) partly inhibited the effect of E2. Besides E2, the partial agonist tamoxifen stimulated PR mRNA expression in ovariectomized rats, whereas the pure antiestrogen ZM 182780 remained inactive in this experiment. Both E2 and tamoxifen caused an autologous down-regulation of ER mRNA. In conclusion, our study demonstrates for the first time that not only the aorta, but also the venous vessels, represented by the vena cava and the portal vein of the rat, are targets for steroid hormones. The ER in vascular tissue is functionally active and mediates direct modulatory effects of estrogens on gene expression in vascular cells.

Estrogen action on hepatic synthesis of angiotensinogen and IGF-I: direct and indirect estrogen effects.

In the present study effects of estrogens (natural estradiol and synthetic ethinyl estradiol) on liver derived proteins (angiotensinogen, IGF-I) were investigated in vivo in ovariectomized rats and in vitro in a rat hepatoma cell line (Fe33). The aim of this study was to establish both an animal and an in vitro model for quantification of the hepatic activity of given estrogenic compounds, and to study underlying mechanisms as regards the question of direct or indirect mode of estrogen action. In ovariectomized rats subcutaneous (s.c.)-treatment for 11 days with either estradiol (E2) or ethinyl estradiol (EE) (dose range 0.1-3 micrograms/animal/day) induced a comparable dose-dependent increase in uterine weight indicating a similar estrogenic potency of the two estrogens. Equipotency was also found as regards the effects on IGF-I plasma levels which dose-dependently decreased by about 50% at the highest dose tested (3 micrograms/animal/day). The decrease in IGF-I serum levels was accompanied by a significant 40% decrease in liver IGF-I mRNA. In contrast angiotensinogen plasma levels were affected only by EE (60% increase for the 3 micrograms/animal/day dose) but not by E2. When rats, in addition to ovariectomy, were also hypophysectomized (substituted with human growth hormone and dexamethasone) angiotensinogen again increased by 80% upon administration of 3 micrograms/animal/day EE, whereas IGF-I remained unaffected by EE. In a rat hepatoma cell line (Fe33) which is stably transfected with an estrogen receptor expression plasmid, 10 nmol/l EE for 24 h caused a 2.4-fold increase in angiotensinogen mRNA level. We conclude from our studies that estrogen effects on angiotensinogen serum levels in the rat are direct effects via the hepatic estrogen receptor, whereas estrogen effects on IGF-I serum levels are indirect effects, the primary target of estrogen action being probably the pituitary. The changes in angiotensinogen serum levels in the rat model are comparable to the situation in humans indicating the rat model and the Fe33 model to be useful tools to study the hepatic activity of estrogenic compounds.

Extracellular matrix induces hormone responsiveness and differentiation in RUCA-I rat endometrial adenocarcinoma cells.

We recently described the establishment and the characterization of two rat endometrial adenocarcinoma cell lines which we called RUCA-I and RUCA-II. Despite fairly high estrogen receptor levels neither cell line responded to estradiol in conventional cell culture conditions on plastic and in the presence of serum. A limited hormonal response to the antiestrogen tamoxifen was detectable in RUCA-I but not in RUCA-II cells. To advance our cell culture conditions we plated RUCA-I cells on a layer of reconstituted basement membrane (Harbor Matrix) in the presence of a serum-free defined medium. These cell culture conditions induced hormone responsiveness of RUCA-I cells and permitted a stimulation of proliferation by estradiol. Further, two estradiol-induced secretory proteins with an apparent molecular weight of 115 kD and 60 kD could be identified by SDS-gelelectrophoresis if analyzed under reducing conditions. These proteins migrated as a single band in a non-reducing electrophoresis gel and were identified as components of the complement C3 system. Additionally, our results suggest that the effects of extracellular matrix and hormones on the expression of these proteins are additive. We conclude that processes of functional differentiation are most likely to occur in this in vitro model, particularly since the expression of components of the complement C3 system was under estrogenic control. Complement C3 proteins represent major estradiol-inducible secretory protein of the immature rat uterus in vivo. Culturing RUCA-I cells on top of a layer of reconstituted basement membrane provides a novel tool to study the importance of the extracellular environment on the hormone-induced gene expression in endometrial carcinogenesis in vitro.

Identification of estrogen regulated genes in Fe33 rat hepatoma cells by differential display polymerase chain reaction and their hormonal regulation in rat liver and uterus.

We applied the differential display RT-PCR (ddRT-PCR) technology to identify estrogen-regulated hepatic genes in the estrogen receptor expressing rat hepatoma cell line Fe33. Three genes of known sequences were detected by the ddRT-PCR approach: IGF binding protein-1 (IGFBP-1), vitamin D-dependent calcium-binding protein (CaBP9k) and major acute phase protein (MAP). Effects of ethinyl estradiol on the mRNA levels of these genes were confirmed by "Northern-blot" analysis. If given in combination with dexamethasone and glucagon, ethinyl estradiol caused 40-, 15- and 11-fold increases in the mRNA steady state level of IGFBP-1, CaBP9k and MAP, respectively, in Fe33 cells 24 h after addition of hormone. Besides ethinyl estradiol, the partial estrogen agonist OH-tamoxifen caused dose dependent effects on expression of MAP and IGFBP-1. Estrogen regulation of the respective genes and the modulatory effects of progesterone (10 mg/animal/day) were studied in ovariectomized rats treated subcutaneously for 14 days with 1 microgram/animal/day estradiol. "Northern-blot" analysis of liver RNA revealed a 6-fold stimulation of IGFBP-1 mRNA levels in estradiol-treated compared to vehicle-treated rats and a weak but detectable increase of MAP mRNA steady state level (1.6-fold) upon estradiol administration. No effect of estradiol treatment could be monitored for CaBP9k in rat liver. Modulatory effects of progesterone on estradiol-stimulated expression in the liver could be monitored for IGFBP-1 only. In an extension of our investigation on the expression of the three genes in rat liver, we determined their expression and hormonal regulation in the uterus of the same animals. In the uterus, estradiol caused an increase in CaBP9k mRNA. In contrast, IGFBP-1 mRNA levels increased dramatically upon progesterone administration, whereas no effect of estradiol treatment could be detected. MAP mRNA levels increased only after coadministration of estradiol and progesterone. In conclusion, the ddRT-PCR proved to be a powerful method to identify estrogen-regulated genes. The study on the hormonal regulation of three genes stimulated by estrogen in Fe33 cells revealed similarities and differences in their regulation in vivo and in vitro.

Cell-specific inhibitory and stimulatory effects of Fos and Jun on transcription activation by nuclear receptors.

We investigated the effect of c-Fos and/or c-Jun co-expression on transcription activation by the progesterone (PR), glucocorticoid (GR) or androgen (AR) receptors using three different reporter genes and four different cell lines. We found that c-Fos could only inhibit, while c-Jun could either inhibit or further stimulate receptor-induced transcription. All these effects were receptor, promoter, and cell type specific, and, importantly, the steroid receptors had non-reciprocal effects on the transactivation ability of c-Jun in the presence or absence of c-Fos. Collectively, these results argue against heterodimer formation as a mechanism to explain the phenomena. Transactivation by the endogenous PR in T47D cells could be inhibited by increasing the intracellular c-Fos level with forskolin as well as by co-expressing c-Fos; no such effect was seen in MCF-7 cells. The inhibition by c-Fos of PR-induced transcription involves a competitive mechanism, which requires the presence of the intact c-Fos leucine zipper and is directed mainly at the transcription activation function (TAF) located in the PR and GR hormone binding domains (TAF-2). However, the co-expression of c-Fos did not alter the 'squelching/transcriptional interference' by the PR of estrogen receptor (ER)-induced transcription. Multiple mechanisms are discussed which may be involved in the crosstalk between the two signal transduction pathways.

Establishment of a human endometrial cell culture system and characterization of its polarized hormone responsive epithelial cells.

Uterine epithelial cells from normal human endometrium were cultured as a primary cell culture in a dual-chambered system. The epithelial cells were isolated from endometrial tissue of the proliferative phase obtained by hysterectomy. The epithelial cells were seeded on Millicell CM filters coated with the extracellular matrix Matrigel. Depending on the culture conditions, the epithelial cells formed a polarized cell monolayer on Matrigel or gland-like structures in Matrigel. The epithelial cell polarity was maintained during culture, which could be proved by electron microscopy. The progesterone and estrogen receptors as typical marker molecules for physiologically intact endometrial epithelial cells could be detected immunohistochemically as well as by RT-PCR in vitro and were down-regulated by medroxyprogesterone acetate (MPA) used as progesterone analogue. As this cell culture system exhibits morphological and immunohistochemical characteristics, typical for the in vivo situation, and since it can be modulated by hormone treatment under the in vitro conditions described, it represents a valuable tool for investigating processes that are essential for endometrial differentiation and reproductive functions.

Identification of selective estrogen receptor modulators by their gene expression fingerprints.

Clinical studies have shown that estrogen replacement therapy (ERT) reduces the incidence and severity of osteoporosis and cardiovascular disease in postmenopausal women. However, long term estrogen treatment also increases the risk of endometrial and breast cancer. The selective estrogen receptor (ER) modulators (SERMs) tamoxifen and raloxifene, cause antagonistic and agonistic responses when bound to the ER. Their predominantly antagonistic actions in the mammary gland form the rationale for their therapeutic utility in estrogen-responsive breast cancer, while their agonistic estrogen-like effects in bone and the cardiovascular system make them candidates for ERT regimens. Of these two SERMs, raloxifene is preferred because it has markedly less uterine-stimulatory activity than either estrogen or tamoxifen. To identify additional SERMs, a method to classify compounds based on differential gene expression modulation was developed. By analysis of 24 different combinations of genes and cells, a selected set of assays that permitted discrimination between estrogen, tamoxifen, raloxifene, and the pure ER antagonist ICI164384 was generated. This assay panel was employed to measure the activity of 38 compounds, and the gene expression fingerprints (GEFs) obtained for each compound were used to classify all compounds into eight groups. The compound's GEF predicted its uterine-stimulatory activity. One group of compounds was evaluated for activity in attenuating bone loss in ovariectomized rats. Most compounds with similar GEFs had similar in vivo activities, thereby suggesting that GEF-based screens could be useful in predicting a compound's in vivo pharmacological profile.