MECHANISMS INVOLVED IN THE REGULATION STEROID RECEPTOR LEVELS

OF

PETER W. JUNGBLUT, ALUN HUGHES, XJRGEN GAUES, ERHARD KALLWEIT’, ITZHAK MASCHLER, FRITZ PARL, WALTER SIERRALTA, PABLO I. SZENDRO and NJDIGER K. WAGNER

Max-~~anck-Institut fiir Zellbiologie, P.O. Box 1009, D-2940 Wilhelmshaven, Germany and ‘Institut fiir Tierzucht und Tierverhalten FAL, Mariensee, D-3057 Neustadt 1, Germany SUMMARY The turnover of steroid receptors comprises: synthesis in the cytoplasm, translo~at~on into the cell nucleus and degradation at a still unknown site. From studies on the oestradiol/receptor system, the following conclusions can be drawn. 1. The in-uioo uptake of oestradiol by target cell nuclei is receptordependent. Steroid and receptor are translocated from the cytoplasm in a 1: f ratio. A recycling of receptor is undetectable after pulse administration of oestradiol. Receptor replenishment in the cytoplasm is accomplished by synthesis. 2. The nuclear uptake of receptor-in contrast-proceeds also in the absence of oestradiol. Both forms of receptor, monomer and “activated” dimer are present in oestrogen-free nuclei. 3. Oestradiol enhances the “nucleotropy” and the turnover rate of receptor. 4. Oestrogenicity and antioestrogenicity are apparently linked to effects exerted on the “nucleotropy” of receptor and its ability to interact with the relevant nuclear structures.

soluble phase of the cytoplasm [Sj, in the nucleus [63 and in the cytoplasmic membranes, constituting the microsomal fractions of homogenates [7]. Before discussing their differences, it should be emphasized that in all three receptors, the steroid-binding core is identical, as judged by the same high affinity and specificity they exhibit towards oestradiol. The distinguishing physico-chemical properties (Fig. l), therefore, must arise from modifications which do not directly involve the steroid-binding site. We characterize the extracted receptors, after invitro saturation with labelled oestradiol and removal of the excess steroid by adsorption to charcoal, by measuring the electrophoretic mobilities and the sedimentation velocities of the complexes formed. Electrophoresis in agar gel at low temperature f8], originally designed as an alternative to the more costly centrifugation in sucrose gradients, proved to be a very rewarding procedure in more than this respect. It led to the discovery of the “basic” microsomal receptor and showed that the “acidic” cytosol and nuclear receptors have the same net charge, despite their state of aggregation.

INTRODUCTION Receptors are known to be essential for the accumulation of steroid hormones inside the ceil nucleus. This aspect of receptor function accentuates the importance of the steroid and suggests a passive rather than an active role for the receptor. In the light of

recent data [l-3], however, the transcription-regulating protein called “receptor”, itself, must be considered as the acting element, the conformation of which is favorably influenced by the steroid. Receptor action can be best studied by following the sequence of events prompted in the “resting” cell by a well-defined stimulus. Our model of choice is the oestrogen/re~ptor system in the pig uterus [4], which offers two advantages: 1. This steroid/receptor system is “self-perpetuating”, i.e. the protein governs the transcription of its own message and the translated product thus is an indicator of action. 2. The gross anatomy of the pig uterus and the mating behaviour of pigs allow for the application of pulsed stimuli, undisturbed by adverse effects elicited by excitement or anesthesia. MATERIALS AND METHODS

The radioactive compounds used in this study were better than 95% pure. All other chemicals were of analytica grade. The experimental procedures have been described in detail in references IF;,8, 16, 171. RESULTS AND DISCU~ION 1. Properties and subcellular distribution of uterine oes-

trogen receptors The presence of oestrogen receptors has “been described in three compartments of the cell: in the 273

The relationship between the classical cytosol “9s” and the nuclear “5s” receptor has been the subject of many investigations ever since their first description. While we initially failed in interconverting the “9s” cytosol receptor-extracted with hypotonic media-and the “5s” nuclear receptor-extracted with buffered Ct.3M KCI solution-by salt exchange [6,9], Korenman and Erdos [IO, I 11 were able to dissociate the large molecule by the addition of salt. Success and failure are apparently a function of time. The fresher the extract at salt treatment, the higher the yield of the dissociation product from the “9s’ and larger aggregates. Their composition is still poorly understood and it is even debatable, whether they actually do exist in-uiuo or are formed during homogenization, extraction and thereafter.

214

PETER

W.

JIJNCBLUT rr d.

cells while preserving

their structural elements. We therefore use two approaches, which should be complementary.

Fig. 1. Physical characteristics of oestradiol receptor from three cellular compartments. LH side-Migration in agargel electrophoresis at low temperature. RH side-Sedimentation profiles following sucrose density gradient centrifugation (gradients for cytosol and nuclear receptor con-

tained 0.3 M KCl). The second striking difference, besides net charge, between the microsomal and the cytosol/nuclear receptor is, that the former entirely lacks the tendency to aggregate. Its sedimentation velocity-in low salt-is slightly slower than that of the salt-dissociated cytosoi receptor. Our then unsuccessful attempts to strip the suspected “‘acidic” and aggregating glycoproteins off their carbohydrate moiety and thus revert them to the “basic”, steroid-binding core protein, uncovered the phenomenon and the mechanism of “oestradiol-mediated” receptor dimerization [12]. Kinetic analyses [13,14,4] of the heat-induced transformation of the cytosol receptor [15] later lent additional support to the hypothesis, that “activation” for intranuclear action involves the formation of a receptor dimer. Accordingly, the cytoplasmic receptars-“basic” microsomal and “acidic” cytosol-are extracted as monomers which can be dimerized in vitro, while the “acidic”” nuclear receptor is commonly a dimer, which can be dissociated into monomers, indistinguishable from the “acidic” cytosol receptor (Fig. 1). The relative ease, with which receptors can be characterized is unfortunately not matched by an equally easy extractability from quantitatively separated subcellular fractions. Uterus is composed of muscle, a layer of connective tissue lined with epithelial cells and interspersed with tubular cells and additionally contains considerable amounts of extracellular collagen, glycosaminoglycans and proteoglycans. This composition gives little chance for a disruption of all

The first approach relies on differences in extractability and physico-chemical properties of the recaptors, solubilized sequentially from the suspension of cell fragments obtained by pulverizing uterine tissue in liquid nitrogen [163. Apparently more than 95% of the “acidic” cytosol receptor is found in the first high speed supernatant (Fig. 2). The successively extracted quantities of “basic” (microsomal) receptor point to its structura1 origin and add up to a figure close to that for the cytosol receptor. This high proportion underlines the hitherto widely overlooked biological significance of the microsomal receptor, most of which is trapped in the low speed sediment of homogenates prepared from fresh tissue. The saltextractions of the ‘*acidic” nuclear receptor also show a reasonable reproducibility, but are far from being exhaustive. For this and other reasons, work is in progress to further improve the procedure. The second approach consists in the analysis of purified nuclei, isolated from unfrozen uterus. Our technique [l?] yields l&20”/, of the total nuclei present and the isolated fraction is a 60:40 mixture of endometrial and myometrial nuclei. By morphometric analysis, debris account for approx. 10% of the pellet volume. An important step in the preparation is the removal of the outer layer of the nuclear envelope, which is part of the rough endoplasmic reti-

Fig. 2. Diagram showing relative amounts of acidic and basic receptors extracted sequentially by the buffer series shown.

Mechanisms involved in the regulation of steroid receptor levels

275

Fig. 3. Basic mechanisms of action of steroid hormones. Circled numbers indicate components, the analysis of which is recommended for the determination of hormone-sensitivity in human breast cancer biopsies. @= Cytosol oestrogen receptor. @= Oestradiol in “nuclear” sediment. @ = Cytosol progesterone receptor. culum and contains “basic” microsomal receptor. Its presence or absence, therefore, is an additional, biochemical criterion for purity. The quantitative extraction of receptor, even from purified, “stripped” nuclei is still an unsolved problem. Although digestion with micrococcal DNAase [18] roughly doubles the yields obtained by the classical salt extraction[16], twice as much receptor again can be extracted after enzymatic breakdown to the core protein (manuscript in preparation). The validity of kinetic experiments, therefore, rests largely on the reproducibility of the extraction procedure employed. 2. Turnover of receptor in uterine cells aj?er stimulation by oestradiol From the analyses of receptor distributions at various times after the intrauterine administration of 20ml of a 1 x 10V6 M solution of oestradiol to chronicaIly castrated pigs, a general scheme of recep tor turnover can be deduced (Fig. 3). Its special features are: (a) the interpretation of receptor-“activation” for nuclear uptake as a dimerization process, (b) the claim, that translocated receptor is being not reshuttled to the cytoplasm for repeated use and consequently, (c) that new “acidic” cytosol receptor arises by synthesis and that the “basic” microsomal receptor represents its core-protein precursor [i, 41. It also demonstrates the seemingly indispensable function of oestradiol for receptor translocation from the cytoplasm into the nucleus and all subsequent events. This then undisputed property of the steroid led to a “logical” suggestion, made by one of us (PWJ) at the 1975 meeting of the International Study Group for Steroid Hormones in Rome: Since the receptor needs the steroid for nuclear entrance-and vice lpersu-the (easier) assay of the steroid in the “nuclear sediments” of breast cancer biopsies, in addition to that of the cytosol receptor, must be a useful supplement for the determination of hormone-responsiveness.

When studying this preposition in the pig uterus model by analyzing the receptor and the oestradiol content of purified nuclei after an oestradiol pulse, it indeed turned out, that oestradiol enters the nucleus together with an equivalent number of binding sites (Fig. 4). But, the concentration patterns for receptor and oestradiol (up to 8 hours) only run in parallel, they do not coincide! The starting level for receptor lies in the range of some 10,000 binding sites per nucleus, that for oestradiol in the range of some 100 molecules. Although the sudden rise in nuclear receptor content is matched by an equally sudden decline in “acidic” cytosol receptor, the concentration of the latter does not begin to increase again, until the number of nuclear binding sites has fallen to and even somewhat below the control value (not shown). Later than 8 h after the oestradiol puIse, the until then parallel course of nuclear oestradiol and receptor content diverges. While oestradiol continues to fade out of the nucleus, the receptor concentration shows an upward trend. A comparison of these biochemical data with the time course of morphological events taking place in the oestradiol-pulsed uterus (Fig. 5) casts some doubt on the notion, that the continuous presence of an “essential” amount of oestradiol is required for the overall reaction [19]. It rather attributes a triggerpulling function to the hormone or even only that of a (very effective) accelerator of a basically hormone-independent process, if the mere presence of (“free”) receptors inside the nucleus is a criterion for activity. Resides these specuhtive aspects, the existence of “unfilled” binding sites undoubtedly depreciates the value of “exchange assays” [203 for the determination of endogeneous hormone in experiments with labelled compounds. 3. Receptor

“nucleotropy”

and receptor

“activation”

The apparent nuclear uptake of receptor without accompanying steroid indicates, that receptor “activa-

276

PETER W. JI?NG~HN~ ef ul.

Fig. 4. Amounts of oestradiol and oestradiol receptor faund in uterine nuclei of ovariectomized pigs at various times alter the intrauterine injection of 20 ml of a 1 x IO 6 M oestradiol solution

before and

(injected

at zero time).

oestrone and the uptake pattern after oestriol administration should be different from that obtained with oestradiol. In striking contrast to this assumption, equimolar pulses with either oestrogen result in a virtually identical, speedy accumulation of receptor inside the nuclei (Fig. 6). The retention patterns, however, are markedly different, especially after the oestrone pulse. The oestront-receptor complexes leave the nucleus almost as quickly as they entered it. At 60 min after the intrauterine injection of oestrone, the nuclear receptor level is again down to the control value. It is of interest to note, that in this particular case a reshuttle of receptor into the cytosol occurs. Uptake of receptor by the nucleus and release from it are mirrored by a depletion and a “replenishment” in the cytosol (not shown).

tion” by the hormone is not indis~nsable for the translocation of the protein from the cytoplasm into the nucleus. “Activation” has been interpreted as the formation of receptor dimers, which readily takes place in-vitro, when receptor-containing cytosols are warmed after prior saturation of the binding sites with oestradiol at low temperature. No dimerization is observed with oestrone-saturated monomers. Oestrial is effective, but to a lesser extent than oestradiol[12-141. Pravided, that the different in-citro efficacy of the 3 oestrogens in facilitating receptor dimerization is a true reflection of the in-&o situation and that only “activated” dimers can penetrate the nuclear envelope, no sudden increase in nuclear receptor concentration should be seen after an intrauterine pulse of

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Fig. 5. Schematic representation af the time course of morphological events in the ovariectomized pig uterus following an intrauterine pulse ol oestradiol. At the indicated time points. cross sections from the lower third of the uterus were examined by light and electron microscopy. Morphometric analysis of epithelial cells was carried out by point counting methods[22]. The changes with time are indicated by the thickness ol the bars.

Mechanisms involved in the regulation of steroid receptor levels

277

Fig. 6. Comparison of nuclear entry and retention of receptor after intrauterine injection of oestradiol, oestriol, oestrone and tamoxifen at zero time. The identical potency of the 3 oestrogens for facilitating the nuclear entry of the receptor, in contrast to their different effects on receptor retention-and dimerization!-signify, that the general term “activation” can no longer be used synonomously for both processes. We, therefore, suggest a distinction between effects exerted on the “nucleotropy” of receptors and on their “activation” for the enhancement of transcription. This “activation” consists most likely in the formation of receptor dimers, which are abie “to slip into place”. It is quite probable, that distant parts of the (“acidic”) receptor molecule are responsible for nucleotropy and dimerization. “Basic” microsomal receptor is not found inside the -nucleus, but its dimerization is governed by the same mechanism and is oestrogenfacilitated in the same fashion as is that of the “acidic” cytosol receptor [12]. The attachment of an (“acidic”) nucleotropic tail to the (“basic”) steroid-binding core appears to be the last step in receptor biosynthesis [P]. 4. Ovarian-independent

and

oestrogen-independent

turnover of receptor

Some years ago, we described two types of ovarianindependent fluctuations of the cytosol receptor levels in several mammalian tissues [Zl]. A seasonal variation was found in uteri of calves and ovariectom~ed pigs and in breast cancer biopsies from postmenopausal women. An apparent circadian rhythm was observed in the oestrogen receptor content of ovariectomized rats. Both phenomena were suspected to be caused by a release of adrenal steroids, which are ~ripherally aromatized. A third type of fluctuation with a periodicity of Si2 days in the uteri of ovariectomized/hypophysectomized rats, however, pointed to a steroid-independent turnover of receptor [4]. The possible influence of the adrenal has been investigated by comparing the oestradiol and the recep-

tor content of uterine nuclei from ovariectomized and from ov~i~tom~ed/adrenalectomized pigs [17]. The up to 1500 molecules of o~tradiol/nucleus found 7 weeks after ovariectomy of the immature pigs, clearly indicate, that extra-ovarian oestrogens are not a “quantitt nkgligeable”. Their adrenal origin could be concluded from a faulty adrenalectomy after which the hypertrophied remnants gave rise to nearly 500 molecules/nucieus, while no oestradiol at all was found in the uterine nuclei of completely adrenafectomized (and ovariectomized) pigs. Although the non-exhaustive salt extraction was used in this study, the receptor content of uterine nuclei after ovariectomy alone exceeded that of their oestradiol con~ntration by ratios of 1.8-10.3. After additional complete adrenalectomy, a substantial number of binding sites could be extracted from the nuclei, in spite of the absence of oestradiol. (It should be stressed here, that while extracted receptor and oestradiol can be assayed with the same sensitivity and accuracy, oniy the steroid can be extracted quantitatively, but not the protein.) Density gradient analyses of extracts, prepared 33 days after adrenalectomy and saturated with labelled oestradiol in the cold, demonstrated the presence of both, receptor dimers and-to a lesser extent-receptor monomers (Fig. 7). Foreclosing the remote possibility, that, after a joint entrance-the retention of oestradiol and receptor in the nucleus differs by more than 33 days, it must be concluded that receptor translocation into the nucleus and receptor dimerization are basically hormone-independent processes. The receptor even seems to be capable of exerting an influence on transcription in the absence of oestradiol, as indicated by the above mentioned fluctuations of uterine cytoplasmic receptor levels in steroid-deficient rats [4] and by the death of cultured cells after receptor-poisoning [2].

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PETER W. JUNGRLUT er u/

4. Little M., Szendro P. 1.. Teran C.. Hughes A. and Jungblut P. W.: Biosynthesis and transformation of microsomal and cytosol estradiol receptors. J. steroid Biochem.

6 (1975) 493-500.

molecule for 5. Toft D. 0. and Gorski J.: A receptor estrogens: isolation from rat uterus and preliminary identification. Proc. mtn. Acad. Sci. U.S.A. 55 (1966) 1574-1581. 6. Jungblut P. W., Hatzel I., De Sombre E. R. and Jensen E. V.: Die oestrogenbindenden Prinzipien der Erfolgsorgane. 18th Coll. Ges. phys. Cher+e (Mosbach) Springer-Verlag Berlin (1967) p. 58-86. Uher “HormonRezeproren”.

15 Fig.

-

3a

ml

7. Presence

of receptor monomers and dimers in nuclear extracts of ovariectomized/adrenalectomized animals prepared at 0°C. Density gradient centrifugation: 5-20”,/, sucrose containing 0.3 M KC], 0.003 M NaN, and buffered with 0.05 M phosphate pH 7.5; 14 h at &2’C; 56,000 rpm (308,000 g,,,): SW 56; LZ-65B.

CONCLUDING

REMARKS

The evidence accumulated calls for a reevaluation of the steroid-receptor relationship with the receptor becoming the focal point rather than the steroid [22]. It is not the function of the receptor to transport the steroid to a site of action inside the nucleus, but it is the function of the steroid to act as a “physical catalyst” (a term used by Elwood Jensen in a 1959 seminar at Northwestern University) on the transcription-regulating protein by enhancing its “nucleotropy” and by stabilizing its active (dimeric) form. The assumption of imbalanced effects, exerted on receptor “nucleotropy”, dimer formation and stability by compounds other than oestradiol, offers a reasonable explanation for the degree of their “oestrogenicity” or “antioestrogenicity”. REFERENCES

1. Jungblut P. W., Gaues J., Hughes A., Kallweit E., Sierralta W., Szendro P. and Wagner R. K.: Activation of transcription-regulating proteins by steroids. J. steroid Biochem. 7 (1976) 1109-l 116. 2. Lippmann M., Bolan G., Monaco M., Pinkus L. and Engel, L: Model systems for the study of estrogen action in tissue culture. Ibid (1976) 1045-1051. 3. McGuire W. L., Zana D. T., Chamness G. C. and Horwitz K. B.: Human breast cancer: biologically active estrogen receptor in the absence of estrogen? Science 1% (1977) 663-664.

7. Little M.. Rosenfeld G. C. and Jungblut P. W.: Cytoplasmic estradiol receptors associated with the microsomal fraction of pig uterus. Hoppe-Seyler’s 2. Physiol. Chem. 353 (1972) 231-242. 8. Wagner R. K.: Characterization and assay of steroid hormone receptors and steroid binding serum proteins by agargel electrophoresis at low temperature. HoppeSeyler’s Z. Phgsiol. Chem. 353 (1972) 1235-1245. 9. Jungblut P.,W.. McCann S.. Giirlich L.. Rosenfeld G. C. and Wagner R. K.: Binding of steroids by tissue proteins: Steroid hormone receptors. Research on Steroids IV (Eds. Finkelstein, Klopper, Cassano. Conti). Pergamon Press, London (1970) pp. 213-232. 10. Korenman S. G. and Rao B. R.: Reversible disaggregation of the cytosol-estrogen binding protein of uterine cytosol. Proc. natn. Acad. Sci. U.S.A. 61 (1968) 1028-1031. of a uterine oestradiol receptor. I I. Erdos T.: Properties Biochem.

Biophys.

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32, (1968)

338-343.

12. Little M., Szendro P. and Jungblut P. W.: Hormonemediated dimerization of microsomal estradiol receptor. Hoppe-Seyler’s Z. Physio/. Chem. 354 (1973) 1599-1610. 13. Nielsen S. and Notides A. C.: Transformation of the rat uterine estrogen receptor after partial purification. Biochem.

biophys.

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381 (1975) 377-383.

14. Yamamoto K. R. and Alberts B.: In vitro conversion of estradiol-receptor protein to its nuclear form: Dependence on hormones and DNA. Proc. natn. Acud. Sci. U.S.A. 69 (1972) 2105-2109. 15. Brecher P. I., Numata M.. DeSombre E. R. and Jensen E. V.: Conversion of uterine 4s estradiol-receptor complex to 5s complex in a soluble system. Fed. Proc. 29 (1970) 249. 16 Jungblut B. J.: Sequential extraction of various forms of estradiol receptor. Acra Endocr. Copenh. Suppl. 215 87 (1978)

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17. Jungblut P. W., Kallweit E., Sierralta W.. Truitt A. J. and Wagner R. K.: The occurrence of steroid-free. “activated” estrogen receptor in target cell nuclei. Hoppe-Seyler’s Z. Physiol. Chem. 359 (1978) 1259-1268. 18. Andre J., Raynaud A. and Rochefort H.: Characterization of the estradiol receptor extracted from nuclei hy 17 (1978) Biochemistry nuclease. micrococcal 3619-3626.

19. Anderson J. N.. Peck E. J. Jr and Clark J. H. Estrogeninduced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei.: Endocrinology 96 (1975) 166167. 20. Anderson J., Clark J. H. and Peck E. J. Jr: Oestrogen and nuclear binding sites. Determination of specific sites by (3H) oestradiol exchange. Biochem. .I. 126 (1972) 561-567.

21. Hughes A., Jacobson H. I., Wagner R. K. and Jungblut P. W.: Ovarian-independent fluctuations of estradiol receptor levels in mammalian tissues. J. CeII. Mol. Endocr.

5 (1976)

379-388.

22. Weibel E. R.: Stereological principles for morphometry in electron microscopy cytology. Inr. Rec. Cyrol. 26 (1969) 235-302.