Glucocorticoid receptor in human embryo fibroblasts. I. Kinetic and physicochemical properties.

The kinetics of dexamethasone binding to L 809 E cell line cytosol have been investigated by means of the protamine sulfate precipitation assay. The KDeq for dexamethasone was 1.1--3.3 nM. Binding was specific for glucocorticoids. The mean association rate constant (k+1) was 8.5 x 10(5) M-1 x min-1 and the dissociation rate constant was 4.6 x 10(-5) min-1 at 0 degrees C. The concentration of binding sites was 0.3 pmol/mg of cytosol protein. Binding kinetics were compatible with a model of positive cooperativity. The receptor sedimented at 7.5--9 S in glycerol gradients. By a combination of calibrated ultracentrifugation and polyacrylamide gel electrophoresis, a Stokes radius of 8.5 nm, a molecular weight of 268 000 daltons and a frictional ratio of 1.8 were determined in low ionic strength conditions. When the cells were incubated with 10 nM [3H]dexamethasone for 1 h, a more than 90% depletion of cytosol receptor and an equivalent accumulation of nuclear dexamethasone--receptor complexes was observed.     

Competitive binding studies with glucocorticoid receptors from rat-thymus cells: differential temperature-dependence of steroid binding.

Competitive steroid-binding studies were performed with intact rat thymus cells and with cytosol preparations at different temperatures using [1,2-3H]dexamethasone as the labelled ligand. Steroids lacking a 17 alpha-hydroxyl group, such as corticosterone, were better able to compete with [1,2-3H]dexamethasone for binding to glucocorticoid receptors at 0 degrees C than compounds containing a 17 alpha-hydroxyl substituent, such as cortisol. At 37 degrees C the reverse was true. This temperature-dependent change in relative affinities appeared to be unrelated to steroid metabolism or receptor activation, and to depend only on the thermodynamic parameters of the steroid--receptor interaction. Relative biological activities for different steroids agree more closely with the relative affinities determined at 37 degrees C than with those determined at lower temperatures.     

In vitro inhibition of aldosterone biosynthesis by 17-hydroxylated steroids in rat adrenal

The in vitro effect of the addition of non-17-hydroxylated and 17-hydroxylated steroids on the production of aldosterone has been studied in the rat. The biosynthesis of deoxycorticosterone, corticosterone, and aldosterone was increased when progesterone or pregnenolone was added to the incubation media, but neither cortisol nor 11-deoxycortisol was produced. The addition of 17α-hydroxyprogesterone or 17α-hydroxypregnenolone to the incubation media resulted in the biosynthesis of cortisol and 11-deoxycortisol by the rat adrenal; concomitantly, a decrease in aldosterone production occurred. However, no decrease in aldosterone production resulted when cortisol and 11-deoxycortisol were added directly to the incubation media. These studies show that neither cortisol nor 11-deoxycortisol inhibits the in vitro biosynthesis of aldosterone and suggest that other mechanisms are involved.     

Assay of activated form of progesterone receptor with ATP-sepharose column chromatography

Biological actions of progesterone are correlated with the ability of the progesterone receptor(PR) to bind to nuclear acceptor sites. Measurement of not only the presence of PR in a tissue but also the amount of that receptor which is able to bind to nuclear acceptor sites is important in predicting tissue response to progesterone. Activation of PR is required for effective binding to chromatin. Since the dextrancoated charcoal assay does not distinguish between an activated and a non-activated receptor, a rapid, relatively simple assay is needed which can account for an activated form of PR. Therefore, ATP-Sepharose column chromatography was tested to identify an activated form of PR. PR in crude uterine cytosol from estrogen-primed immature rabbits was labeled with 3H-progesterone at 0 degree C. The labeled PR was then incubated at 4 degrees C with uterine chromatin from ovariectomized mature rabbits. The freshly prepared PR had little capacity to bind to the chromatin. After activation manipulation at low temperature, low ionic strength and neutral pH, this PR was able to bind to chromatin approximately fourfold more than that activated by heating at 25 degrees C. The affinity of the activated and the non-activated PR for ATP was evaluated on ATP-Sepharose column chromatography. The activated PR was selectively adsorbed onto columns of ATP-Sepharose, and the binding ability of the activated PR to ATP paralleled that of the rabbit uterine chromatin. These results suggest that ATP-Sepharose column chromatography could be useful to identify an activated PR as a substitute for chromatin binding assay.     

Progestin receptors in human uterine cytosol

Progestin(norethindrone and norethindrone acetate)-binding protein, exhibiting characteristics similar to uterine progesterone receptor, has been identified in human uterine cytosol. The progestin receptor was characterized by sedimentation coefficient 4.2 S; Stokes radius, 39 Å; frictional ratio 1.29; isoelectric pH 4.6; molecular radius 2.7 nm; and molecualr weight in the range 67 000–74 000. The ammonium-sulfate-precipitated progestin-receptor complex was eluted from a DEAE-cellulose column at 0.18 M KC1. The progestin binding was saturable and stereospecific. The sequential variation in receptor concentration (early proliferative, 3800–4300 sites/cell; late proliferative, 9500–11200 sites/cell; early secretory, 4900–6200 sites/cell; late secretory, 1800–2300 sites/cell) was in conformity for progesterone and the progestins, when concurrently measured. Oral administration of norethindrone significantly reduced the cytoplasmic and nuclear receptor concentration for estradiol and progesterone. A significant observation was that the progestins stabilized the progestin receptor by forming a slowly dissociating complex with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～ 110?130}\text{min}$ as compared with the progesteronereceptor complex dissociating with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～41}\text{min}$. Thus, the uterine progestin receptor recognizes progestins in general, although with a varying degree of affinity, and the altered rate constants could be of putative importance in determining the biological potency of the progestins.     

Changes in net charge of glucocorticoid receptors by activation, and evidence for a biphasic activation kinetics

The kinetics of glucocorticoid receptor activation and the changes in molecular properties of the receptors by the activation were studied, employing aqueous two-phase partitioning of rat thymocyte, rat liver and mouse S49.1 lymphoma cell cytosol labelled with tritiated glucocorticoid. By a mathematical analysis of the time-course of the receptor partition coefficient during activation, we demonstrate that at least two different receptor conversions take place during this process. Partitionings at conditions excluding receptor aggregation allowed an evaluation of differences in net charge between activated and non-activated forms of native and chymotrypsin-treated receptors. The net charge of the chymotrypsinized receptor changes little by the activation, being between 0 and -10 at pH 8 both in the non-activated and the activated state. In contrast, the activation changes the net charge of the native receptor from around -50 to around -10.     

Transformation of a rabbit progesterone receptor from an 8S form to 5.5S and 4S forms

The 8S form of the rabbit uterine progesterone receptor transforms slowly at 0°C to a 4S form with an intermediate 5.5S form. The transformation is accelerated by either heat, increased ionic strength and dilution of cytosol. The transformation, which is reversibly inhibited by sodium molybdate, is unrelated to total cytosolic alkaline phosphatase activity. The transformation is accompanied by a positive change in receptor surface charge and a decrease in the rate of progesterone dissociation. The stability of the 8S progesterone-receptor complex is reduced and the sedimentation coefficient increased by acidic conditions; acid does not affect the 4S receptor as drastically.     

Characterization of different forms of the androgen receptor.

Three forms of the androgen receptor have been characterized at high ionic strength in partially purified cytosol and nuclear fractions of rat ventral prostate, epididymis, testis, and seminal vesicle by ion exchange chromatography on phosphocellulose, gel filtration in Sephadex G-200, and sedimentation in sucrose gradients. The three forms have the following properties, respectively: elution from phosphocellulose at 0.15--0.20, 0.3--0.5, and 0.20--0.32 M KCl; gel filtration radii of 53, 36, and 22 A; sedimentation coefficients of 5.0S, 3.6S, and 3.0S; frictional ratios of 1.65, 1.4, and 1.0; and molecular weights of 115,000, 55,000, and 29,000. In testis and epididymis cytosol, the 53 A, 5S form was more abundant than the 36 A, 3.6S form. In ventral prostate, the 36 A, 3.6S receptor was the predominant form. Variable amounts of all three forms were observed in seminal vesicles. Conversion from 36 A, 3.6S to 22 A, 3.0S was induced by heating ventral prostate cytosol and could be blocked by serine protease inhibitors. The 22 A, 3.0S receptor fragment from heated prostate cytosol was similar in size and symmetry to receptor extracted in 0.5 M KCl from prostate nuclei labeled in vivo. Extracts of epididymis and seminal vesicle nuclei contained both 36 and 22 A forms.     

Progestins inhibit fsh-stimulated steroidogenesis in cultured rat granulosa cells

It has been hypothesized that progesterone (P) exerts a direct inhibitory effect on ovarian follicular development, an effect which could be mediated by P receptors located in granulosa cells. We tested this hypothesis by examining the effect of several progestins on FSH-stimulated estrogen (E), P, and 20α-dihydroprogesterone (DHP) production by cultured rat granulosa cells, and correlated the results with the ability of the progestins to bind to the granulosa cell P receptor. Granulosa cells from immature hypophysectomized DES-treated rats produced 9 ng/ml E, 21 ng/ml P and 29 ng/ml DHP during a 2-day incubation in McCoy's 5a medium containing 10^?7 M androstenedione and 10 ng/ml oFSH. The FSH-induced increase in E production was inhibited by 50 and 95% following concomitant treatment with 3 × 10^?6 and 10^?5 M resp. of R5020, a potent synthetic progestin. Added R5020 at these concentrations also significantly inhibited P and DHP production. R5020 had no effect on granulosa cell viability or plating efficiency, and the inhibitory action of R5020 on E production was reversible. In studies of the specificity of the progestin inhibitory action, the relative abilities of various progestins to inhibit E production were: R5020 > P > DHP > 17α-hydroxyprogesterone (170HP). The relative abilities of these progestins to bind to the ovary P receptor were also: R5020 > P > DHP > 170HP. These results indicate that exogenous progestins directly inhibit the FSH-stimulation of granulosa cell steroidogenesis in vitro and suggest that the progestin effect may be mediated by the P receptor. Such results offer a possible mechanism whereby progesterone could exert a direct but reversible inhibitory action on ovarian follicular development.     

Differentiation between steroid hormone receptors CBG and shbg in human target organ extracts by a single-step assay

The steroid complexes of (plasma) corticosteroid-binding globulin can be distinguished from intracellular steroid-receptor complexes by agar electrophoresis at low temperature in neuraminidase-treated tissue extracts. With this method, the presence of progesterone receptor has been demonstrated in heavily plasma-protein-contaminated human uterus “cytosol”, but not in human mammary carcinoma extracts. SHBG and “basic” receptors for estradiol and dihydrotestosterone in human uterus cytosol could also be assayed simultaneously.     

Chick oviduct progesterone receptor: structure, immunology, function

Analysis of the purified chick oviduct progesterone receptor using biochemical and immunological approaches indicates that while the 'activated' receptor ('4S') is a mixture of two progestin-binding polypeptides, 'A' (Mr approximately 79 kDa) and 'B' (Mr approximately 110 kDa), the non-activated receptor ('8S') is a population of complexes containing a hormone-binding polypeptide (A or B, but probably not both) bound to a non-hormone-binding protein (Mr approximately 90 kDa). Two molecules of the 90 kDa protein appear to be present in each '8S' receptor molecule. The 90 kDa protein is also associated with the non-activated forms of receptors of other steroid hormones in the chick. Molybdate stabilizes the non-activated receptors, probably by forming weak coordination bonds with radicals provided by the subunits of the '8S' structure. Activation implies separation of the subunits, without a change in their primary structure, and does not require intervention of any protein other than those present in the '8S' receptor form. The presence of ligand at the binding site accelerates the activation process but, in vitro, is not necessary for it to occur. Unlike the non-activated form, activated receptors bind to the cell nuclei. However, histological studies with anti-progesterone receptor antibodies indicate that in the non-hormone-exposed tissue the (non-activated) receptors could be localized in the nuclei.     

The impact of radioimmunoassay on our understanding of human adrenal physiology

Radioimmunoassay has only been used for a relatively short time to study the human hypothalamic-pituitary-adrenal system, but the findings have already contributed immeasurably toward a better understanding of the physiology of this system. In some instances classical concepts have been confirmed and extended. Other findings have led to revisions of old concepts and the formulation of new ones. These physiologic studies have produced new and better laboratory tests for the pituitary-adrenal function that are simple and reliable enough to be used not only in clinical research but in the routine practice of medicine. Even greater contributions from radioimmunoassay can be expected in the future.     

Glucocorticoid-induced cell-size changes and nuclear fragility in rat thymocytes

The events preceding glucocorticoid-induced lymphocytolysis have been studied in isolated rat thymocytes. Incubation of thymocytes at 37°C in the presence of 1 μM dexamethasone resulted in the progressive appearance of pyknotic cells of modal diameter 4.6 μm, distinct from normal cells of diameter 5.2 μm. The rate of appearance of the pyknotic cells was determined by selective electronic cell counting, and was shown to be accompanied by increased nuclear fragility. The production of pyknotic cells was glucocorticoid-specific, dose-dependent, blocked by cycloheximide, and preceded the loss of cell viability as determined by dye exclusion. The pyknotic cells were separated from the non-pyknotic cells by density-gradient centrifugation and shown to be solely responsible for the observed nuclear fragility.     

Subunit dissociation and activation of wild-type and mutant glucocorticoid receptors

Apparent molecular weights of wild-type and nti ('increased nuclear transfer') mutant glucocorticoid receptors were obtained from Stokes radii and sedimentation coefficients. At low salt concentrations molecular forms of Mr 328,000 and 298,000 of the wild-type and mutant, respectively, were predominant. Increasing ionic strength resulted in receptor dissociation. Dissociated forms of Mr 130,000 and 63,000 of the wild-type and mutant, respectively, were obtained at 300 mM KCl and above. Some metal oxi-anions prevented dissociation. Receptor activation to allow DNA binding produced the dissociated forms which could be separated from non-activated receptors by filtration through DNA-cellulose or by DEAE-cellulose chromatography. Non-activated wild-type and nti receptors eluted from DEAE-cellulose under identical conditions while activated wild-type and nti receptors eluted differently. Partially proteolyzed wild-type receptors behaved identically to nti receptors. We conclude that the large forms of wild-type and nti receptors are heteromeric and contain only one hormone-building polypeptide per complex.     

Characterization of the calf uterine androgen receptor and its activation to the deoxyribonucleic acid-binding state

The androgen receptor (AR) from calf uterine cytosol has been studied in terms of steroid-binding affinity, hormone dissociation kinetics, and DNA-cellulose-binding capacity. The binding affinity for three androgens, analyzed under conditions where binding to progesterone receptor did not occur, decreased in the order: methyltrienolone greater than 5 alpha-dihydrotestosterone greater than testosterone. Activation of the receptor to the DNA-binding state involved the following changes of the receptor: decrease in dissociation rate for the steroid, disaggregation of the receptor, and increase in affinity for DNA. Dissociation studies with methyltrienolone and 5 alpha-dihydrotestosterone revealed that the AR can exist in two affinity states which differ 13- to 30-fold in their affinity for the steroid. Molybdate (10-20 mM) prevented the formation of the high affinity state. The high affinity state receptor was formed in the absence of molybdate or after ammonium sulfate precipitation (0-40% saturation) of the molybdate-stabilized low affinity state receptor. During formation of the high affinity state, the sedimentation coefficient of the receptor in low ionic strength buffer decreased from 8-9S to 4.5S, indicating receptor disaggregation. DNA-cellulose binding capacity increased from 3 to 65% upon formation of the high affinity state. The DNA-binding form could be eluted from DNA-cellulose at 0.14 M NaCl. After elution the DNA-binding form maintained its sedimentation coefficient of 4.5S and chromatographed as a protein with a Stokes radius of 44 A. From these results it can be concluded that the activated, DNA-binding form of the AR in calf uterus is a protein with a molecular mass of approximately 85,000, which acquires a higher affinity for the ligand upon its formation.     

Progestins inhibit FSH-stimulated granulosa estrogen production at a post-cAMP site

The purpose of this investigation is to examine the mechanism by which progestins inhibit FSH-induced estrogen (E) production by cultured rat ovary granulosa cells. We have demonstrated that the highly potent synthetic progestin, R5020, is able to inhibit the induction of granulosa cell aromatase activity by cholera toxin, prostaglandin E2, dibutyryl cAMP or oFSH. Since the induction of E synthesis by these compounds is mediated through activation of adenylate cyclase and increased cellular cAMP production, these observations indicate that the progestin inhibitory effect is a post-cAMP event. In addition, we have demonstrated that R5020 does not inhibit FSH-stimulated granulosa cell cAMP production. The involvement of the granulosa cell progesterone (P) receptor as a mediator of this post-cAMP progestin effect is suggested by the relative abilities of various progestins to both bind the P receptor and to block the induction of granulosa cell aromatase activity by dibutyryl cAMP. While the precise mechanism of progestin action remains unclear, the kinetic analysis of aromatase enzyme activity demonstrates that progestins are not acting as competitive inhibitors of granulosa cell aromatase. Since E is necessary for follicular development, our in vitro data are consistent with the hypothesis that P is a factor which can inhibit FSH-induced follicular growth and development in the rat ovary.     

Aqueous two-phase partition studies of glucocorticoid receptors exposed to limited trypsination

In a previous investigation the properties of glucocorticoid receptors exposed to partial proteolysis by chymotrypsin were studied by aqueous two-phase partitioning (Andreasen, P.A. and Gehring U. (1981) Eur. J. Biochem. 120, 443–449). This paper describes studies of the properties of cytosolic glucocorticoid receptors submitted to limited trypsination, employing phase partitioning of rat thymocyte cytosol labelled with tritiated triamcinolone acetonide.Trypsin treatment of labelled cytosol at 0°C does not result in any dissociation of steroid from the receptor. The partition properties of the trypsin-treated receptors exposed to receptor-activating conditions are indistinguishable from those of the activated native and chymotrypsin-treated receptors, although the trypsin-treated receptors have lost the affinity for DNA and dextran sulphate. Trypsin treatment of cytosol not exposed to receptor-activating conditions results in a rapid change in the receptor partition coefficients identical to that following chymotrypsin treatment. However, during incubations under conditions at which activation of native and chymotrypsin-treated receptors is very slow, the trypsin-treated receptor is converted to a form with partition properties indistinguishable from those of the activated receptors. During exposure of the cytosol to activating conditions, the time-course of the partition coefficient of the trypsin-treated receptors is only slightly different from that of the native and chymotrypsin-treated receptors, but the trypsin-treated receptors are far less susceptible to the activation inhibitors ATP, Li⁺ and MoO^2?₄.We conclude that the proteolytic cleavages induced by trypsin in the non-activated receptor do not lead to any immediate changes in the charge and surface properties of the receptor different from those following chymotrypsin treatment, but that the trypsin-treated receptor is not able to maintain a non-activated state and a normal susceptibility to activation inhibitors.     

Interaction of glucocorticoid hormones with rat skeletal muscle: catabolic effects and hormone binding.

The mechanism of action of glucocorticoid hormones on rat skeletal muscle was studied by following their effect on muscle weight, free amino acid content, activity of amino acid-metabolizing enzymes, and binding to cytoplasmic receptor proteins. A significant reduction of gastrocnemius muscle and body weight occurred following administration of cortisol, triamcinolone diacetate, and triamcinolone acetonide to adrenalectomized rats. Treatment with triamcinolone diacetate also reduced the level of several free amino acids and enhanced the activity of a myofibrillar protease in skeletal muscle. The hormone had, however, no effect on the activity of various enzymes involved in amino acid catabolism in muscle. In nephrosis, another condition of muscle wasting, the level of several muscle amino acids were also reduced to a lesser extent. Cortisol and triamcinolone acetonide, both of which induce muscle wasting, were found to bind to two distinct cytoplasmic proteins in muscle. Binding of the labeled hormones was followed at 0 C and could be observed in presence of a 1000-fold excess of the catabolically inactive steroid epicortisol. Binding of 3H-triamcinolone acetonide. In vitro competition experiments further suggest a correlation between steroid binding to the 3H-dexamethasone or 3H-triamcinolone acetonide site and their potency to induce muscle catabolism. It is concluded that skeletal muscle is a direct target organ for glucocorticoids, and that muscle responsiveness involves binding of the active hormones to cytoplasmic receptor sites.     

Dissociation between cortisol-induced pycnosis and inhibition of [3H]uridine incorporation in rat thymocytes.

Treatment of rat thymocytes with cortisol induced an inhibition of [3H]uridine incorporation after 30-90 min, an accumulation of pycnotic cells after 90 min, and a decrease in cell viability after several hours. No cortisol-resistant cells could be distinguished, and dose-response curves for a number of glucocorticoids showed a correlation to the saturation of the glucocorticoid receptors. The pycnotic effect of cortisol increased between pH 5.2--7.0 in parallel with a stimulation of the spontaneous development of pycnotic cells. The cortisol-induced accumulation of pycnotic cells and inhibition of [3H]uridine incorporation varied independently as a function of the cell density, and in a glucose-salt medium only the pycnotic effect of cortisol became inhibited. The inhibition of [3H] uridine incorporation is therefore not an integral part of the pycnotic change of the cells. The glucocorticoid sensitivity was found to increase with the age of the animals, before the onset of thymus involution.     

Oestrogen and progestogen receptors in endometrium and myometrium at the time of blastocyst implantation in pregnant diabetic rats

Summary A suitable hormonal environment is a prerequisite for blastocyst implantation. Experimental diabetes was previously shown to modify the hormonal milieu and produce alterations in oestrogen receptor kinetics in the uterine tissue. In the present work, oestrogen and progestogen receptor levels were measured on the morning of day 6 of pregnancy in normal and in streptozotocin-induced diabetic rats, both in implantation sites and in interembryonic segments of endometrium and myometrium. Receptor levels were different in the implantation sites compared to the interembryonic segments of endometrium, both in the control and in the diabetic animals. Indeed, implantation sites were characterized by lower oestrogen receptor levels in cytosol and higher progestogen receptor levels in cytosol and nuclei. However, compared to the control rats, the diabetic rats had lower oestrogen receptor levels in implantation sites, both in cytosol and nuclei. In the myometrium, the differences between sites or between types of rats were minimal. Plasma levels of oestradiol were lower in diabetic rats than in control animals, whereas progesterone levels were similar. A 20% lower implantation rate was found in diabetic rats, compared to normal rats. These results show that the specific distribution of oestrogen and progestogen receptors between implantation sites and interembryonic segments was preserved in the diabetic rats; however the absolute level of oestrogen receptor was lower. This abnormal endocrine milieu might arise from a lower oestradiol level and a decreased oestradiol/progesterone ratio in the circulating blood. Whether the lower implantation rate in diabetic rats might be a consequence of the overall disturbed hormonal status remains to be elucidated.