The non-DNA-binding heterooligomeric form of mammalian steroid hormone receptors contains a hsp90-bound 59-kilodalton protein

Untransformed cytosol receptors for progesterone (PR), androgen (AR), estrogen (ER), and glucocorticosteroid (GR) in rabbit tissues contain a 59-kDa protein (p59) (Tai, P.K.K., Maeda, Y., Nakao, K., Wakim, N.G., Duhring, J.L., and Faber, L.E. (1986) Biochemistry 25, 5269-5275) and a 90-kDa heat shock protein (hsp90). In the present study, receptors from calf uterus (PR, AR, ER, and GR) and from human breast cancer MCF7 cells (PR and GR) were also shown to be comprised of hsp90 and p59. These heterooligomer receptor complexes were stabilized both by transition metal oxyanions (molybdate and tungstate) and chemical cross-linking with dimethylpimelimidate. In 0.4 M KCl, tungstate-stabilized (but not molybdate-stabilized) PR, AR, ER, and GR retained hsp90, but lost p59. Dimethylpimelimidate cross-linking prevented p59 dissociation from hsp90-receptor complexes. Stabilization with tungstate and/or cross-linking permitted immunoaffinity purification of untransformed rabbit as well as calf PR and ER on EC1-Affi-Gel 10 column (an anti-p59 immunoadsorbant). Combined immunoaffinity purification and cross-linking experiments indicated that p59 is bound to hsp90 in the cytosol. We propose that in the nontransformed steroid receptor, p59 interacts with hsp90 rather than with the hormone binding subunit.     

The nonactivated progesterone receptor is a nuclear heterooligomer

The discovery of the nuclear localization of estradiol and progesterone receptors in the absence of the steroid hormone has led to reconsideration of the model of cytoplasmic to nuclear translocation of these receptors upon exposure to hormone. Unoccupied nonactivated receptors are thought to be weakly bound to nuclei of target cells from which they are leaking during tissue fractionation and thus found in the cytosol fraction of homogenates in a nontransformed heterooligomeric "8-9 S" form, which includes hsp90. However, no direct biochemical evidence has yet been obtained for the presence of such heterooligomers in the target cell nucleus, possibly because it dissociates in high ionic strength medium used for extraction of the nuclear receptor. We took advantage of the combined stabilizing effects of tungstate ions and antiprogestin RU486 to extract a nuclear non-DNA binding nontransformed 8.5 S-RU486-progesterone receptor complex from estradiol-treated immature rabbit uterine explants incubated with the antagonist. As demonstrated by immunological criteria and by irreversible cross-linking with dimethylpimelimidate, the complex contained, in addition to the hormone binding unit, hsp90, and p59, another nonhormone binding protein. Control experiments carried out with the progestin R5020 yielded the expected nuclear transformed DNA binding 4.5 S-R5020-progesterone receptor complex. These results offer evidence for two distinct forms of steroid receptor in target cell nuclei. Besides the classical "4 S" agonist-receptor complex, tightly bound to the DNA-chromatin structure and in all probability able to trigger the hormonal response, we have observed in the RU486-bound state a non-DNA binding nontransformed 8.5 S form, presumably already present in the nucleus in the absence of hormone and representing the native nonactive form of the receptor.     

Testosterone-induced meiotic maturation of Xenopus laevis oocytes: evidence for an early effect in the synergistic action of insulin

Meiosis reinitiation in oocytes (stage 5-6 of Dumont) isolated free of follicle cells by collagenase treatment from ovarian pieces of Xenopus laevis, was studied in observing the germinal vesicle breakdown (GVBD) provoked by progesterone and testosterone (0.1 nM-1 microM), alone or in association with insulin (30 micrograms/ml). Testosterone, was more active than progesterone to elicit GVBD in vitro, raising the question of the relative roles of both steroids in the physiological maturation process in vivo. The potentiating effect of insulin, already observed on progesterone action, was also demonstrated upon testosterone effect; the results suggested that it occurs during the early phase of hormone-induced meiosis reinitiation.     

From the structure of steroid receptors to their assessment by immunocytochemistry in target cells

Immunohistochemical studies with antibodies to steroid hormone receptors provide new insight in the mechanism of action of steroid hormones. Immunologically reactive estrogen and progesterone receptors are found exclusively in cell nuclei of target cells even in the absence of the hormonal ligand. A hormonal treatment inducing receptor transformation and "translocation" to the nucleus does not modify the intracellular distribution of the receptor. This result is in contradiction with most biochemical studies which show a displacement of receptor from the cytosolic fraction to the nuclear fraction after hormone-receptor complex formation. We propose that different affinity levels of the non-transformed and hormone-complexed receptor molecules for nuclear structure produce unequal losses of nuclear receptor during homogenization. A lesser loss appears as an increase in nuclear binding sites or immunologically reactive receptor. The glucocorticosteroid receptor differs from the others in that it shows an increase of nuclear immunoreactive receptor after hormone administration. This result was accepted as evidence for a nuclear translocation in the sense initially proposed for all steroid hormones. Alternatively, one may propose another explanation based on the same experimental artefact as invoked for the estrogen and progesterone cytosol receptors. A higher affinity of the hormone-complexed receptor entails a lesser loss from the nucleus during tissue processing, and consequently an apparent increase in nuclear staining. Such a possibility is currently tested in parallel with the progesterone receptor.     

The antiprogesterone RU486 stabilizes the heterooligomeric, non-DNA-binding, 8S-form of the rabbit uterus cytosol progesterone receptor

The salt-induced (0.3 M KCl) transformation of the non-transformed, heterooligomeric 8S-form of the rabbit uterus cytosol progesterone receptor (PR) was analyzed by density gradient ultracentrifugation (8S----4S conversion) and DNA-cellulose chromatography (non-binding----binding forms). After 1 h treatment at 2 C, greater than 90% of agonist (R5020 or Org2058)-PR complexes were transformed, contrary to antagonist (RU486)-PR complexes, which did not undergo any transformation. Thus, there is stabilization of the non-transformed receptor form by RU486 as compared to the effect of agonist binding. The hydrodynamic parameters of both agonist- and antagonist-bound non-transformed receptors were similar and the calculated Mr were approximately 283,000 and approximately 293,000, respectively. In both cases, purification indicated the presence of a 90-kD non-hormone-binding protein associated with the hormone binding unit(s). Transformation of RU486-PR complexes occurred after exposure to high salt at increased temperature and was correlated to the dissociation of the 90-kD protein from the receptor. Both agonist- and antagonist-bound transformed forms of PR had apparent similar affinities for DNA-cellulose. Molybdate-stabilized and KCl-treated RU486-PR complexes were more stable, as assessed by steroid binding, than the corresponding R5020-PR complexes, arguing in favor of a stabilizing effect of both the 90-kD protein and RU486 against inactivation. These cell-free experiments support the concept that RU486 in the rabbit uterus system stabilizes the 8S non-DNA binding, non-transformed form of the receptor at low temperature. The possibility that impaired dissociation of the heterooligomeric receptor form is involved in the antiprogesterone activity of RU486 is discussed.     

Steroid hormone radioautography in cultured cells: Studies with Shionogi carcinoma 115

A radioautographical technique for the localization of soluble compounds in cultured cells is described. It has been used to investigate the distribution of steroid hormones in target cells, and an example is given with the Shionogi carcinoma SC-115, an androgen-sensitive mammary tumor in mice. Experiments at 37 °C have given direct evidence for the specific binding of [³H]-androstanolone (5α-dihydrotestosterone) in the cytoplasm. This result is based on the labeling after incubation with 0.5 nM radioactive androgen, on the isotope dilution-test showing a limited capacity, and on competition assays with other hormones. Results also show the transfer to and the specific binding of the hormone in the nucleus. No cellular labeling has been observed at 0 °C, even when using 10 nM of high specific activity [³H]androstanolone. The technique appears promising for the study of various aspects of the interactions between hormones and isolated cells.     

Cell-specific variations and hormonal regulation of immunoreactive cytochrome P-450scc in the rat ovary

Specific rabbit antibodies to the bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc) were used to cross-react with the enzyme in the rat ovary. The luteal cells of cyclic, pregnant, and pseudopregnant rats were immunostained. P-450scc was also expressed in the interstitial cells of prepubertal and cyclic adult rats, and in the thecal cells lining the preovulatory follicles. In cyclic females, RU 486 and oestradiol increased the intensity of P-450scc immunostaining. The granulosa cells of ovarian follicles whatever their stage of development, including preovulatory follicles, were not labelled, except after ovulation. The intensity of immunostaining of thecal and interstitial cells decreased during early pregnancy or pseudopregnancy, and disappeared after Day 9, whereas these cells were intensely labelled 24 h after parturition. The immunostaining of thecal and interstitial cells was again detected in 18-day pregnant rats, treated with the antiprogesterone RU 486. It is therefore concluded that both oestradiol and progesterone are involved in P-450scc regulation.