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Abstract
3,5,3′-Tri-iodothyronine (T3), 1α,25(OH)2-vitamin D3 (D3) and retinoids activate related nuclear receptors which interact by heterodimerisation to regulate gene expression. Actions of each hormone are discrete and may be specified by changes in the relative concentrations of their receptors (T3R, vitamin D receptor (VDR), retinoic acid receptor (RAR), retinoid X receptor (RXR)). T3, D3 and retinoids are essential for skeletal development and maintenance and we have previously shown complex interactions amongst their signalling pathways in osteosarcoma cells. In these studies we demonstrate that similar T3R, VDR, RAR and RXR proteins are co-expressed in both osteoblast lineage cell primary cultures and osteosarcoma cells by Western blotting. We investigated whether hormone interactions in bone result from changes in receptor stoichiometry. Cells were treated with combinations of T3, D3, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (RA) that are known from previous studies to produce complex cell specific responses. No alteration in expression of any receptor protein was seen in response to any hormone combination in three phenotypically distinct osteosarcoma cell lines. Thus, in contrast to studies of overexpressed receptors in vitro, changes in the physiological concentrations of endogenous T3R, VDR, RAR and RXR do not specify discrete hormone actions in osteoblastic cells. Other unidentified factors are likely to modulate hormone action in these bone cells.
Journal of Endocrinology (1997) 154, 63–74
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ABSTRACT
This paper describes the effect of oestradiol-17β implants on unbound cytosolic and nuclear oestrogen receptors in the uterus and anterior pituitary gland of ovariectomized adult rats. Rats were ovariectomized and implanted 1 week later with oil or oestradiol-17β oil solution in silicone elastomer capsules. In the latter animals the capsules either remained in situ until decapitation (subgroup 1) or were removed 48 h after implantation (subgroup 2). Implants of oestradiol-17β caused a significant depletion in both forms of oestrogen receptor in the uterus and anterior pituitary gland of rats in subgroup 1. In subgroup 2, however, there was an almost complete return to control concentrations of uterine cytosolic receptors and anterior pituitary cytosolic and nuclear receptors. Concentrations of uterine nuclear oestrogen receptors showed only a partial recovery.
These data suggest that both forms of oestrogen receptor constitute an integrated population of oestrogen receptors, without dependence on intracellular location, and that in the presence of oestradiol these receptors are bound more quickly than they are synthesized. The results also indicate the existence of a dynamic equilibrium of unbound receptors between the cytosolic and nuclear compartments for both target organs, but show that in the absence of oestradiol this equilibrium is restored in the anterior pituitary sooner than in the uterus.
J. Endocr. (1987) 115, 205–210
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SUMMARY
The concentration of progesterone receptors in rat uterine cytosol and nuclei was measured during the oestrous cycle and pregnancy. The method used allowed the measurement of the total concentration of binding sites (unbound and hormone-bound).
During the oestrous cycle, the concentration of receptors in the cytosol peaked at pro-oestrus, low concentrations were observed at oestrus and metoestrus, and an increase was seen at dioestrus. In the nuclei, maximum concentrations occurred at pro-oestrus and dioestrus. The number of receptors in the cytosol was very low during the first half of pregnancy, but the concentration increased progressively after day 15 to attain a very high level (about 26 000 binding sites/cell) on day 22.
In the nuclei, the concentration of receptors was low at the beginning of pregnancy. On day 5 (day of implantation) there was a slight increase, which corresponded to a decrease in the number of cytosolic receptors and a small peak in the level of progesterone in the plasma. Maximum concentrations were attained during a 'plateau' period between days 9 and 15. Thereafter, there was a decrease in the concentration of nuclear receptors and on day 22, the mean value was very low; in some animals, probably on the verge of parturition, no receptors were detectable in the nuclei.
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Specific oestradiol binding to a receptor in nuclear and cytosol fractions of the rat anterior pituitary gland and pituitary responsiveness to gonadotrophin releasing hormone (GnRH) during the oestrous cycle have been studied. To accomplish this, both unoccupied and occupied oestradiol-binding sites in the cytosol and oestradiol-binding sites in the nucleus and total cell were measured during the oestrous cycle. The concentration of unoccupied and occupied sites and total oestradiol binding in the cytosol fluctuated during the cycle. At pro-oestrus, the concentration of cytosol receptor was diminished by about 40% and replenishment occurred during oestrus. On the other hand, a profound increase in concentrations of cellular and nuclear receptors occurred at pro-oestrus. Administration of GnRH significantly stimulated LH release at all stages of the cycle. The maximum stimulation of LH release by GnRH was observed at 13.00 h of pro-oestrus. From these studies, it is concluded that pituitary responsiveness to exogenous GnRH during pro-oestrus parallels the changes in the content of oestrogen receptors in the cytosol and nucleus.
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SUMMARY
Measurement of the uptake and retention of a radioactive post-coital antifertility agent tamoxifen, by reproductive tissues of the rat have shown that the ovary retained more radioactivity than did any other reproductive organ. Studies have also been made of the uptake and distribution of [3H]tamoxifen and [3H]oestradiol-17β in the uterus of the pregnant rat on days 2–6 post coitum. Twenty-four hours after administration of tamoxifen, either i.v. or orally, 40–50% of the radioactivity was in the high speed pellet, 10–20% in the nuclear fraction, and 15–30% in the cytosol. An equivalent dose of [3H]oestradiol-17β yielded distributions of 5%, 5% and 82% respectively. Fractionation of uteri from animals given 0·2 mg tamoxifen/kg on Day 2 of pregnancy followed by [3H]oestradiol 60 min before death showed little difference in total uptake of oestradiol or distribution in the subcellular fraction on Days 4, 5 and 6. Although uptake of oestradiol by uterine nuclei was reduced on Day 3 by previous administration of tamoxifen on Day 2, appreciable quantities were still bound to the nuclear receptors. Treatment of ovariectomized animals with tamoxifen at doses up to 40 μg/rat (i.e. 0·2 mg/kg) led to the accumulation of oestrogen–receptor complex in the nucleus.
It is concluded that the antifertility properties of tamoxifen (under the conditions of these experiments) cannot be ascribed to the suppression of uptake and binding of oestradiol by the uterus.
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SUMMARY
The uptake of [6,7-3H]oestradiol in vivo and in vitro by cell fractions from regions of rat brain and the anterior pituitary gland has been studied. Cytoplasmic and nuclear receptors were detected in anterior and posterior hypothalamus but not in brain cortex.
After labelling in vivo, tissues took up [6,7-3H]oestradiol in the following order of magnitude: anterior pituitary > anterior hypothalamus > posterior hypothalamus > cortex. With the exception of the cortex, all extracts from mature tissues had a higher uptake/mg protein than did extracts from immature animals. In the in-vitro system, oestradiol-17β competed with [6,7-3H]oestradiol-17β in the hypothalamus whereas progesterone, testosterone and oestradiol-17α did not. In the pituitary, oestradiol-17α and 17β competed for binding sites.
A single injection of testosterone propionate on the second day of life affected [6,7-3H]oestradiol binding in later life. By 28 days of age, the androgenized animals had a lower nuclear and higher cytoplasmic uptake of [6,7-3H]oestradiol in anterior hypothalamus. This effect was not seen in the posterior hypothalamus or cortex. Binding was decreased in all fractions from the pituitary. In mature animals (60 days old), binding fell in both nuclear and cytoplasmic fractions from anterior hypothalamus and pituitary. The nuclei from posterior hypothalamus also took up less [6,7-3H]oestradiol after androgenization. Androgenization affected specific binding in uteri at both 28 and 60 days of age.
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Departamento de Ciencias Químicas y Fisiológicas, Sede Santiago Sur, Universidad de Chile, Casilla 3931, Santiago, Chile
CONTENTS
PAGE
Introduction 287
Pre-transcriptional aspects of the mechanism of action of androgens 288
Binding of androgens to plasma proteins 288
The metabolism of androgens in the androgen-sensitive tissues 290
The binding of androgens to cytoplasmic receptors 291
Prostate 292
Kidney 293
Muscle 293
Bone marrow 294
Skin 294
Urogenital tract of the embryo 294
The binding of androgens to other cytoplasmic components 295
The nuclear binding of androgens 296
Nuclear receptor sites 296
Nuclear acceptor sites 298
Transcriptional and post-transcriptional aspects of the mechanism of action of androgens 299
Action on chromatin template capacity and activity 299
Action on RNA polymerase 301
Action on biosynthesis and pool size of RNA precursors 302
Action on RNA maturation 302
Action of androgens on protein biosynthesis 303
Action of androgens on DNA synthesis 304
Some comments on the effect of androgens on transcriptional and post-transcriptional events 304
General remarks 305
References
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Introduction
Steroid hormones bound to their specific nuclear receptors exert diverse physiological effects on gene expression in target tissues. One important mechanism by which cells modify hormone action is by modulating the structure of the steroid hormone ligand. 17β-Hydroxysteroid dehydrogenase (17β-HSD) is an enzyme involved in the activation, reducing 17-keto steroids to 17β-hydroxy steroids, and inactivation, oxidizing 17β-hydroxy steroids to 17-keto steroids, of androgens and estrogens (Fig. 1). Its reductase activity increases the affinity of the cognate receptors for their ligands, whereas 17β-hydroxy steroids are inactivated by the dehydrogenase activity. A general observation is that gonadal tissues convert 17-keto steroids to 17β-hydroxy steroids, where the opposite is observed for extragonadal tissues. It is unlikely that the redox potential of a given cell determines the direction of the reaction. More likely is that differential expression of 17β-HSD isozymes determines the net activity in a given tissue. The recent cloning of multiple
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The thyromimetic compound SK&F L-94901 shows more potent thyromimetic activity in the liver than in the pituitary gland or heart when administered to rats. The mechanisms of liver-selectivity of SK&F L-94901 were examined using cultured rat hepatoma cells (dRLH-84) and rat pituitary tumor cells (GH3), both of which showed saturable cellular uptake of tri-iodothyronine (T(3)). When isolated nuclei with partial disruption of the outer nuclear membrane were used, SK L-94901 competed for [(125)I]T(3) binding to nuclear receptors almost equally in dRLH-84 and GH3 cells. SK L-94901 also did not discriminate thyroid hormone receptors (TR) alpha1 and beta1 in terms of binding affinity and activation of the thyroid hormone responsive element. In intact cells, however, SK L-94901 was a more potent inhibitor of nuclear [(125)I]T(3) binding in dRLH-84 cells than in GH3 cells at an early phase of the nuclear uptake process and after binding equilibrium. These data suggest that SK L-94901 is more effectively transported to nuclear TRs in hepatic cells than in pituitary cells and therefore shows liver-selective thyromimetic activity. In conclusion, SK L-94901 discriminates hepatic cells and pituitary cells at the nuclear transport process. The cellular transporters responsible for this discrimination were not evident.
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Some plant compounds or herb mixtures are popular alternatives to conventional therapies and contain organic compounds that bind to some nuclear receptors, such as the estrogen receptor (ER), to exert various biological effects. We studied the effect of various herbal extracts on ERalpha and ERbeta isoforms. One herbal extract, Rhei rhizoma (rhubarb), acts as an agonist to both ERalpha and ERbeta. The phytochemical lindleyin, a major component of rhubarb, might contribute to this estrogenic activity through ERalpha and ERbeta. 4-Hydroxytamoxifen, an ER antagonist, completely reversed the estrogenic activity of lindleyin. Lindleyin binds to ERalpha in vitro, as demonstrated using a fluorescent polarization assay. The in vivo effect of rhubarb extract was studied using a vitellogenin assay system in the freshwater fish, Japanese medaka (Oryzias latipes). There were marked increases in serum vitellogenin levels in male medaka exposed to rhubarb extract. We conclude that lindleyin, a component of some herbal medicines, is a novel phytoestrogen and might trigger many of the biological responses evoked by the physiological estrogens.
