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Multiple forms of nuclear binding of glucocorticoid-receptor complexes in rat thymocytes
Journul of Srerord Bmhumi~fn, Vol. 13, pp. 105 to I12 Pergamon Press Ltd 1980. Printed m Great Britain
MULTIPLE FORMS OF NUCLEAR BINDING OF GLUCOCORTICOID-RECEPTOR COMPLEXES IN RAT THYMOCYTES JOHN A. CIDLOWSKI* and ALLAN MUNCK Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire, U.S.A. (Recriurd 18 April 1979) SUMMARY Exposure of isolated rat thymocytes to glucocorticoids at 37°C leads to localization of 50~-80~ of the o$ular glucocorticoid-receptors on or within the nucleus. In this study, we have used differential salt extraction of nuclei from cells incubated with glucocorticoids under various conditions to further characterize the nature of the nuclear glucocorticoid-receptor interaction. Of the dexamethasone initially bound to nuclei, 70-75% is resistant to 0.2 M KC1 salt extraction and 2$_30% is resistant to 0.4 M KC1 salt extraction. These two resistant fractions are designated N, and N, respectively. The N, fraction contains N4 since Nz can be further extracted with 0.4 M KCl, whereas N4 is resistant to further extraction with either 0.2 or 0.4 M KCl. These data suggest that Na and the difference N2_.&= N, - N& (i.e. that fraction which is resistant to 0.2 M but not to 0.4 M KC?) represent distinct forms of nuclear dexamethasone-receptor interaction. In intact cells at 37°C both N2_., and N, are formed at all the steroid concentrations, and neither fraction shows evidence of saturation. The two fractions differ, however, in the following ways: (a) N4 is formed prior to N,_, under conditions where the rate of formation of the cytoplasmic dexamethasone-receptor complex is not limiting; (b) the addition of a cold chase of unlabeled dexamethasone to cells pre-exposed to [‘HI-dexamethasone decreases N,_, after 60 min by about 70”/, but N, by only 10-15%; (c) even at concentrations of unlabeled dexamethasone which do not decrease total cellular binding a cold chase causes a sharp reduction in N,_, but not in N,; (d) N, decreases more rapidly than N,_, following a decrease in hormone concentration by dilution; (e) the magnitude of the dilution determines the extent of N4 reduction, and little or no change occurs over the 35-90 fold range; (f) cold chases with the anti-glucocorticoids cortexolone and progesterone significantly decrease N2_&but had little effect on N,. The distribution of Rluc~orticoid-re~ptor complexes in the N,_, and N, fractions depends on the steroid which is bound to the receptor. Triamcinoione acetonide,which has similar in r&o biological potency to dexamethasone, has a similar N4 fraction (29%) of nuclear receptor-bound steroid but a greater N,_, fraction (60%). Cortisol, which is 10% as active as dexamethasone, has an N4 fraction of only 13% and an Ns_.+ fraction of 41%. These data indicate that glucocorticoids form physiologicaily distinct classes of nuclear acceptor-receptor complexes.
INTRODUC’HON
Rat thymic lymphocytes respond to physiological levels of glucocorticoids with rapid alterations in cellular metabolism and contain receptors with high affinity and specificity for both natural and synthetic glucocorticoids [l-4]. Hormone-receptor complexes formed in the cytoplasm are rapidly translocated to the nucleus at 37°C and under steady-state conditions most of the receptor-bound steroid is in the nucleus [Z, 43. Since most of the known metabolic responses appear to require RNA synthesis [3], the nucleus seems a likely target for the initiation of responses to glucocorticoids. Recently, considerable effort has been directed at determining the nature of nuclear hormone-receptor binding sites for all classes * Present Address: Department of Biochemistry, University of Vermont. Dexamethasone = (9 a-fluoro-I 1& 17, 2l-trihydroxy-~6ff methylpregna-1,4,-diene,-3,20-dione): triamcinolone acetonide = (9 a-fluoro-1 I/I’, I&X, 17x, 21, tetrahydroxy pregna1,4-diene-3, 20 dione-16, 17-acetonide); cortisol = (I Is, 17a, 21 trihydroxy-4 pregnene-3, 20 dione). 5.8.
13/2--n
of steroid-receptor complexes [ 5- 143. DNA [S, lo], nuclear acidic proteins [6,7,9, 1l] and nuclear membranes Cl43 all have been suggested as possible sites. The approach used in most of these investigations has been to expose cells in uiuo or in culture to a single con~ntration of radioactive hormone, then to isolate nuclei and chromatin, and determine the hormone bound to chromatin subfractions. Studying the actions of estradiol on rat uterus, Clark et al.[lS] have taken an alternate approach using differentia1 salt extractions. They have found that long-term retention of hormone in a fraction of nucleic that is unextractable by 0.4 M KC1 correlates well with uterine growth. Similar high-salt residual nuclear fractions were originally observed with glucocorticoids in hepatoma cells [ 16,171 and subsequently in cultured fibroblasts [18], the latter studies indicating that the salt-residual form of nuclear glucocorticoid receptors increases with energy deprivation. In the present studies we have taken a physiological approach to the characterization of different nuclear forms of glucocorticoids-receptor complexes in 105
JOHN
106
A. CIDL~WSKI
thymus cells, measuring the distribution and kinetic behavior of salt-extractable and unextractable nuclear complexes in intact cells exposed to various conditions which mimic those to which cells are exposed in the whole organism. We present evidence that thymocytes contain at least two distinguishable classes of nuclear glucocorticoid-receptor complexes which associate in unique manners with nuclei. The distribution of these classes of glucocorticoid receptors within whole cells is dependent on steroid concentration, length of time of hormone exposure and biological potency of the steroid molecule. METHODS General
Thymus tissue was obtained from male SpragueDawley rats that had been adrenalectomized 6-8 days prior to.sacrifice and mainlined on 0.9% NaCl and rat chow. Thymus cell suspensions were prepared in Krebs-Ringer Bicarbonate Buffer with lOmM glucose (KRBG) equilibrated with 95% oxygen; 5% CO1 as described previously [I]. The cytocrit (packed cells per ml. of cell suspension) of each preparation was adjusted to 0.2-0.3. Unlabeled dexamethasone, cortexolone and progesterone, purchased from Steraloids, were prepared in concentrated stock solutions ( 10e4 M) in KRBG, and were added to cell suspensions to give the concentrations indicated. [6,7-‘HIDexamethasone (33.0 Ci/mmol), [ 1,23H]-cortisol (45.8 Ci/mmol) and ~4,73H]-tri~cinolone acetonide (33.7 Ci/mmol) were obtained from New England Nuclear. Aliquots of solutions of radioactive steroids dissolved in 9w; benzene, 10% ethanol were placed in the incubation vials, and the organic solvents evaporated to dryness prior to the addition of cell suspensions. The concentrations of steroids given in the results are the total average concentrations. The effective concentrations of free steroid to which the cells are exposed are lower because a substantial fraction of the steroid is bound, most of it nonspecifically [I]. Approximate factors by which the total concentrations should be multiplied to give the free concentrations, taking into account the cytocrit of the cell sus~nsion [i] are: dexamethasone, 0.4; cortexolone, 0.6; progesterone, 0.13. fncubatiun of thymus ceils with steroid and measurement ofcytoplasmic and nuclear receptor binding Suspensions of thymus cells were incubated with tritiated steroids at l-2 x lo-* M in the presence or absence of unlabeled dexamethasone 2 x 10m6M. Receptor-bound tritiated steroid was determined as the difference between values obtained without and with unlabeled hormone. The cell suspensions (generally 300-500~1) were incubated in 1 dram screw-cap glass vials or lOm1 Erlenmeyer flasks which were flushed with 95% 0,:5x COz. The vials were shaken at 20 cycles per minute in a water bath maintained 37°C. Cytoplasmic binding was measured by placing
and ALLAN MUNCK
a 20~1 aliquot of the thymus cell suspension into lOO$ of dextran-coated charcoal in 1.5 mM MgCl, at O”C, vortexing the mixture, leaving it for 15 min at 3°C and sedimenting the charcoal and cellular debris at 15,OOOgfor 2min [19]. Samples of the supernatant were then removed for assessment of radioactivity. Except where a different procedure is described in the figure caption, nuclear binding was measured by placing a 20~1 aliquot of thymus cell suspension into 15 ml polyethylene tubes containing lOm1 of 1.5 mM MgCf, at O”C, incubating this suspension for 15 min at 3”C, sedimenting the nuclei by centrifugation at 3000 g for 5 min. The supernatants were decanted and the tips of each tube containing the nuclear pellet cut off and placed into a liquid ~intillation vial[i9]. Both cytoplasmic and nuclear samples were counted for radioactivity in 5.0ml of Brays solution using a Packard Tri-Carb liquid scintillation counter with efficiency of 25% for tritium. Salt extraction procedures Nuclear pellets from the cells in 20~1 of a suspension that had been incubated with tritiated steroid alone or tritiated steroid plus unlabeled dexamethasone were prepared by breaking the cells in lOm1 of 1.5mM MgCll and centrifuging as described above. These nuclei were then extracted with 500$ of ice cold 1.5 mM MgCl, (control), 0.1 M KCI, 0.2 M KCl, 0.3 M KCl, 0.4 M KC1 or 100% ethanol in the same 15 ml polyethylene tube used in the preparation of the nuclei. After addition of each solution, the pellets were vortexed vigorously for 10s and placed on a Thomas rotating apparatus at speed setting No. 7-8 (60 cycles/min) for 30min at 3°C; under these conditions the pH of the extraction solutions was approximately seven. The pellets were then immediately centrifuged at 3OOOg for 5 min, and lOO/*l of the supernatants taken for measurement of radioactivity. The remaining solution and pellet were diluted by adding lOm1 of 1.5 mM MgCl, at 0°C followed immediately by centrifugation at 3000g for 5 min, the supernatants decanted and the tips of each tube containing the residual nuclear pellet cut off and placed in liquid ~ntil~tion viafs for counting. These procedures give a measure of both extracted and residual nuclear-bound radioactivity. The conditions of these extractions yielded maximal extraction and were independent of the amount of nuclei used, up to nuclei from 100 /.d of cell suspension. All results are given as the difference between the bound cpm from the incubations without and with added unlabeled dexamethasone. RESULTS
Nuclear distribution of glucocorticoid receptors in rat tjymocytes Figure 1 shows a salt-extraction pattern of receptor-bound dexamethasone from thymocyte nuclei. Control nuclei were extracted with 1.5 mM
. L
Heterogeneity of nuclear glucocorticoid-receptor
,cpm
Cpm
E300 51 .z Q E s 200 8 -z 2 m
KCI (M)
Fig. I. Salt extraction pattern of dexamethasone-receptor from nuclei of cells exposed to [“HI-dexamethasone. Suspensions of thymus cells were incubated with [“HI-dexamethasone with or without dexamethasone in Erlenmeyer flasks for 30min at 37°C as described in Methods. Nuclei were prepared and extracted as outlined in the Methods. The data shown represent residual nuclear radioactivity values obtained from a typical experiment. Extractions were carried out in quadruplicate. Results are the means f standard errors obtained after subtracting the non-saturable counts in the presence of unlabeled dexamethasone.
MgClz in order to assess steroid dissociation during the 3°C extraction procedure. Less than 5% of the steroid dissociated from the receptor under these conditions. Of the dexamethasone receptor initially bound, about looO/, is resistant to 0.1 M KCI, 7&75x is resistant to 0.2 M KC1 and 25% is resistant to 0.4 M KCI. Higher salt concentrations (0.5 and 0.6 M KCI) did not lead to any further extraction and caused the nuclei to form a jelly-like mass. For subsequent results, receptor binding resistant to extraction by a particular KC1 concentration is designated as follows: N, is the fraction resistant to extraction by 0.1 M KCl, N, is the fraction resistant to extraction with 0.2 M KCl, etc. When the extracts of nuclei treated with 0.4 M KC1 were exposed to dextran-coated charcoal approximately 50% df the total extracted radioactivity was not absorbed and so was presumably in the steroid-receptor complex form. All of the nuclearbound dexamethasone was extracted following treatment of the nuclei with lOOo/,ethanol. In each experiment we have also measured the amount of radioactivity extracted; we find excellent correlation between total initial binding, extracted steroidreceptor complex and the resistant fractions. For simplicity, we show only the resistant fraction. Similar salt extraction patterns of nuclear glucocorticoid receptor complex have been observed for hepatoma cells [ 163, and fibroblasts [ 183. The data shown in Fig. 2 demonstrate that only extraction solutions of higher ionic strength are effective in removing nuclear-bound dexamethasone resistant to previous salt extraction. A second extraction with 0.1 M or 0.2 M KC1 does not decrease the N2 or N4 fractions while second extraction with 0.3 M or 0.4M KCl, however, decreases the N, fraction down to the N4 level. These studies suggest three points: (a) the first extractions are maximally effective at each
107
interactions
1
q
of Nq
b 100 h J B
OF&fore t IO.1 IO.2 IO.3 Reextraction Clont roi KCI (M)
f0.4,IEtO
.
id
Fig. 2. Re-extraction of N2 and N4 fractions of nuclearbound dexamethasone-receptor. Suspensions of thymus cells were incubated as for the experiments in Fig. I. Nuclei were prepared and extracted with either 0.2 M KCI or 0.4 M KC1 as described in Methods. The pellets were then resuspended in 10 ml 1.5 mM MgClz at 0°C and the suspensions immediately centrifuged at 3000 g for 5 min. The supernatants were discarded and some of the pellets containing N2 and N4 fractions were used for measuring radioactivity (values designated before) and the rest were extracted a second time with 0.1 M KCI, 0.2 M KCI, 0.3 M KCl, 0.4M KC1 or lOOo/,ethanol. The values shown are mean of quadruplicate measurements. Error on these determinations was not greater than 5% on different samples of nuclei.
salt concentration; (b) the N, fraction is contained in the N, fraction, or in other words, the fraction resistant to 0.4 M KCI is also resistant to 0.2 M KCl, but not vice-versa; (c) the salt extraction procedure defines two more-or-less discrete fractions, N, which is resistant to 0.4 M KCl, and the difference N,_, = N2 - N4 which is resistant to 0.2 M KC1 but not to 0.4M KCl. Our subsequent results are expressed in terms of Nz. N*, the calculated N2_-4and the amount bound to unextracted whole nuclei. The data in Fig. 3 shows the dependence of these fractions on [‘HI-dexamethasone concentration. N,, N, and N,_, are present at every concentration in roughly the same proportions (except at the lowest concentrations, as noted below). Half maximal values occur at about 10 nM, the K, for binding of dexamethasone to the receptors, suggesting that the nuclear sites do not become saturated. There is, however, a slight indication of preferential occupation of the N4 sites at low hormone concentrations, below 10nM. We have obtained this result consistently in several experiments. Figure 4 shows the time-course of occupation of the N2_4 and N4 fractions after dexamethasone is added to cells. The N4 fraction increases rapidly and reaches a plateau by 3 min, then continues to increase slowly. The N, and NzA fractions increase for about 15 min before leveling out. Under the conditions of the experiment in Fig. 4, the overall rate-limiting step appears to be formation of cytoplasmic steroid-receptor complex [ 1,4]. A sep-
108
JOHN A CIDLOWSKI~II~ ALLAN
OL Dexamethasone
Concentration (nM)
Fig. 3. Influence of [3H]-dexamethasone concentration on the distribution of the nuclear-bound dexamethasone fractions. Suspensions of thymus cells were incubated with various concentrations of C3H]-dexamethasone with or without unlabeled dexamethasone for 30 min at 37°C. Nuclei from 20~1 of the cell suspension were prepared and either not extracted (whole nuclei) or extracted with 0.2 M and 0.4 M KC1 for determination of N, and N, fractions
MUNCK
carried out this experiment twice, with the same result. We have recently found that the addition of a “chase” of unlabeled dexamethasonone to cells previously equilibrated with [3H]-dexamethasone leads to a decrease in nuclear binding of C3H]-dexamethasone that is considerably faster than the decrease in cytoplasmic binding [4]. The experiments in Fig. 6 examine the kinetics of a cold chase of unlabeled dexamethasone on the rate of loss of the N, and N,-, fractions. Total nuclear dexamethasone decreases rapidly with 60”/, of the initially bound hormone lost after 60 min. During this time, NzP4 decreases in proportion to the total nuclear binding, whereas N, is reduced only marginally. In Fig. 7 we show the magnitudes of the various nuclear fractions 1 h after chase at 37°C with several concentrations of dexamethasone. Here the N,_, fraction again is displaced to a much greater degree than the N4 fraction. A particularly striking observation is
as described m Methods. Values represent the means of at least four separate extractions on different aliquots of cells. N 2m4were calculated as N, - N,.
2000
.
1500
. I'
arate experiment was therefore designed to measure formation of N, and N2_-4 under conditions where the rate of activation of steroid-receptor complex is ratelimiting [2,4]. Cytoplasmic steroid-receptor complex was formed by incubation of whole cells with steroid at 0°C for about 3 h. Using a procedure developed previously [2], the cells were warmed rapidly and kept at that temperature for the number of seconds indicated in Fig. 5. The data shown in Fig. 5 show that the N4 fraction begins to be formed immediately on warming, but the N,_, fraction is formed very slightly if at all. This experiment indicates that the N, fraction is formed prior to the N,_, fraction. We have
:
l:
500-
Dexomethasone
Fig. 4. Time-course of formation of N,, N4 and N,_, fractions after addition of dexamethasone to thymus cells at 37°C. Suspensions of thymus cells were incubated with [3H]-dexamethasone with or without unlabeled dexamethasone at 37°C for the time period indicated. The reactions were stopped by pipetting 20~1 of cells into IOml of ice cold I .5 mM MgCl,, followed by preparation of nuclei and measurement of the N2 and N4 fractions as described in Methods. Each value is the mean of four separate determinations. N,-, = N, - N,.
lW
"'
NUCLEI
0
20 SECONDS
After
.
lti
WHOLE
.
%
0
Mknutes
.
. . .
40 WARMED
80
60 TO
37”
C
Fig. 5. Time-course of formation of N,, N, and N,-, fractions after cells with preformed dexamethasoneereceptor complexes are warmed to 37°C. Suspensions of thymus cells were incubated with [‘HI-dexamethasone with or without unlabeled dexamethasone for 18c-240 min at 0°C. Triplicate 20 ~1 aliquots were removed and added to 200 ~1 KRBG at 37°C in 15 ml conical polyethylene centrifuge tubes with the tips in a 37°C water bath. For the O-s time points the cells were placed in 200~1 KRBG at 0°C. After the indicated number of seconds the cells were simultaneously cooled and disrupted by addition of 1Oml 1.5 mM MgCI, at 0°C. The resulting suspensions were centrifuged to obtain nuclear pellets. One of these was used to obtain counts for whole nuclei, and the other two were extracted to determine the N, and N, fractions, and N 2-4 = N, - N,. Each point is the result from one pellet, corrected for saturable binding.
Heterogeneity
of nuclear
glucocorticoid-receptor
109
interactions
7
Whole
Nuclel
After
Dilution
9) 100 i
o- $S&.-:;
IO Minutes Minutes
After Dexamethasone Chase
Fig. 6. Time-course of loss of nuclear-bound [‘HI-dexamethasone fractions following a cold chase of unlabeled dexamethasone. Suspensions of thymus cells were incubated with [3H]-dexamethasone, with or without unlabeled steroid for 30min at 37°C. Twenty-PI aliquots of cell suspension were removed for measurement of the whole nuclear binding and the N, and N, fractions. Unlabeled dexamethasone was subsequently added to each group to give a final concentration of 2 PM, and the incubations continued. At the intervals indicated, samples of cell suspension were removed for measurement of total nuclear binding, and the N4 and N2 fractions. The values are mean values of four separate extractions from different aliquots of cells. N,_, = N, - N.,.
that at low concentrations of unlabeled dexamethasone (3 x lo-’ M) which hardly affect whole nuclear dexamethasone or N,, there are significant decreases in the N,_, fraction. The next experiments (Fig. 8) examine the influence of removal of the [3H]-dexamethasone on the timecourse of loss of total nuclear dexamethasone binding and of the N,_, and N, fractions. Thymus cells pre-
Fig. 7. The influence of chases with unlabeled dexamethasone at various concentrations on nuclear binding after 1 h. Ten groups of cell suspensions, 200 ~1 each, were incubated with [‘HI-dexamethasone with or without dexamethasone for 30 min at 37°C. Unlabeled dexamethasone in 5 ~1 ahquots at concentrations from 10e4 M to 6.25 x 10e6 M, was added to each group to give the final unlabeled dexamethasone concentrations indicated. The incubations were continued at 37°C for an additional 60min. Total nuclear dexamethasone-receptor binding and the N, and N, fraction were measured in nuclei from four separate 20~1 aliquots of cells. Each point represents the mean value for residual binding. N,_, = N, - N,.
20
3(
Fig. 8. The influence of removal of [3H]-dexamethasone on the time-course of loss of nuclear dexamethasonereceptor. Two l.Oml aliquots of thymus cell suspensions were equilibrated with C3H]-dexamethasone with or without unlabeled dexamethasone for 30 min at 37°C and 20 ~1 aliquots were removed for measurement of total nuclear dexamethasone-receptor and the N, and N, fractions. Twenty ~1 aliquots of cell suspension were placed into 2.0ml of KRBG at 37°C and allowed to incubate for the periods of time indicated. The dilution reaction was stopped at each time interval by adding 8.0ml of ice cold 1.5 mM MgCI,, followed by immediate centrifugation at 3000 g for 5 min. The supernatant of each tube was then decanted, nuclei were prepared by resuspending the pellet in 1.5 mM MgCI,, etc, and total nuclear dexamethasonee receptor and N, and N, fractions were measured. N 2-4 = N, - N,.
viously incubated with [3H]-dexamethasone with or without unlabeled hormone were diluted lOO-fold into KRBG for the intervals of time indicated. Total nuclear binding and the N, fraction rapidly decrease after dilution with little measurable N4 remaining in the nuclei after 30min. Surprisingly in view of the results from the chase experiments, there is little if any decrease in the N,_, fraction. These data show that the N, fraction is more sensitive than the N,_, fraction to reductions in C3H]-dexamethasone con-
Dllutton
Fig. 9. The influence of the magnitude of dilution on the decrease in nuclear bound fraction. Cell suspensions were incubated with [3H]-dexamethasone with or without unlabeled dexamethasone as described in Methods. Twenty ~1 aliquots of cell suspension were then diluted into different volumes of KRBG at 37°C with the dilution ratios indicated. All groups of cells were then allowed to incubate for an additional 30 min at 37°C. The dilution reactions were stopped by the addition of 1.5 mM MgCI, at 0°C and the cell pellets were processed as described in Fig. 8. Values are the mean of determinations from three to four different aliquots of cells.
110
JOHN A.
CIDLOWSKIand ALLANMUNCK
Table I. Distribution of nuclear bound-steroid receptor complexes for cortisol, dexamethasone and triamcinolone acetonide
Steroid Dexamethasone Triamcinolone Cortisol
Nuclear bound steroid Total (moU 0 2.6 x IO-l4 5.2 x IO-“’ 0.5 x lo-l4
80 90 54
2 29 29 14
%ii 50 61 40
Thymus cells were incubated with 2.0 x IO-* M of each steroid with or without 4 x 10e6 M unlabeled dexamethasone for 30 min at 37°C. Total nuclear binding, N, binding and N, binding was then measured in nuclei from 20~1 aliquots of cell suspensions. Residual binding values are the mean of two separate experiments. N,_, = N, - Nq. centration. Similar results have been obtained with [3H]-cortisol and [3H]-trimcinolone acetonide (Cid-
lowski and Munck, unpublished). Figure 9 shows how the magnitude of the reduction in whole nuclear binding and the NzA and N* fractions depends on the extent of reduction of hormone concentration by dilution. Dilution of cells by lOOfold for 30min at 37°C reduces the N4 fraction by 95% and the N,_, by 55%. Dilutions of lesser magnitude cause smaller total reduction in nuclear binding but always lead to a greater loss of N4 than N2_& As shown in Table 1, following exposure of thymocytes to equal molar concentrations of dexamethasone, cortisol and triamcinolone acetonide, unequal amounts of steroid are associated in the nuclei. These values correlate well with the affinities of these
Minutes
After Chase
Cortexolone
Fig. 10. The influence of cold chase of unlabeled cortexolone on the time-course of loss of nuclear dexamethasonereceptor. One ml aliquots of thymus cell suspensions were equilibrated with [3H]-dexamethasone with or without unlabeled dexamethasone for 30 min at 37°C and 20~1 samples of cell suspension removed for measurement of each nuclear binding fraction. To the remaining cell suspension, lOpI of 10m4M unlabeled cortexolone in KRBG was added to give a final concentration of 10e6 M, and the incubation continued. Total nuclear binding, N, and N4 nuclear binding were measured in nuclei from 20~1 aliquots of cell suspension removed at the times indicated. The values shown are then mean f standard error from determinations of four separate samples at each time point. N,_, = N, - N.,.
00 Minutes
15
30
After Chase
45
6C
Progesterone
Fig. Il. The influence of a cold chase of unlabeled progesterone on the time-course of loss of nuclear dexamethasone-receptor. One ml aliquots of cell suspensions were equilibrated with [“HI-dexamethasone as described and total nuclear binding and the N, and N4 fractions measured in nuclei from 20~1 aliquots. To the remaining cell suspension 40 ~1 of 2.0 x IO-’ M unlabeled progesterone prepared in KRBG was added to give a final concentration of 8 x IO-‘M and the incubation of cell suspension continued. After the indicated times, 2O/.d aliquots of cells were removed for assessment of nuclear binding. Each value represents the mean f standard error of quadruplicate determinations. N2_4 = N2 - N4..
for the cytoplasmic glucocorticoid receptor [3]. As observed earlier (Fig. l), nuclear dexamethasone-receptor complexes are relatively resistant to 0.2M KC1 extraction (N2), as are those for nuclear triamcinolone acetonide-receptor complexes. However, only 54% of the initially bound cortisol-receptor complexes are resistant to 0.2 M KCl. Cortisol, dexamethasone and triamcinolone acetonide receptor complexes all have similar N2_4 fractions (4&61x) and dexamethasone and triamcinolone acetonide, two steroids with equal in vitro biological activity (4), have similar N4 binding (29%). The N, fraction for the weaker glucocorticoid, cortisol, is about half that of either synthetic compound. Thus, the natural and synthetic glucocorticoids form different types of nuclear complexes. Figures 10 and 11 respectively, show the results on nuclear-bound [3H]-dexamethasone of chases with cortexolone and progesterone. Both these steroids are known to have anti-glucocorticoid activity [3]. Both of these steroids decrease total nuclear binding of [‘HI-dexamethasone after 60min. During the first 15 min, before whole nuclear binding has decreased, cortexolone initiates a rapid reduction in the NZ_, fraction with little alteration in the N4 fraction. The chase with progesterone is less effective than with cortexolone, probably due to the lower free progesterone concentration used (see Methods). The data in Fig. I1 show, however, that unlabeled progesterone is also capable of causing significant reductions in whole nuclear binding and in the NZ_., fraction, but fails to significantly alter the N4 fraction. steroids
Heterogeneity of nuclear glucocorticoid-receptor interactions DISCUSSION
Several investigators studying the mechanism of steroid hormone action have suggested that heterogeneity exists in the nature of steroid-receptor complexes [8,1 l-143. These suggestions were prompted by observations that maximal physiological responses could be obtained by exposure of cells to concentrations of steroid which would not occupy all available cytoplasmic steroid receptors, and that subpopulations of nuclear steroid-receptor complexes show differential susceptibility to extraction with KC1 [S, 151. Using isolated thymocytes, a system with which one can conveniently obtain nuclei from cells treated with a variety of hormonal conditions, we have sought to probe the interactions of glucoeorticoidreceptor complexes with nuclei. Our studies demonstrate (Figs 1 and 2) the presence of at least two different populations of glucocorticoid-receptor complexes within or on thymocyte nuclei. These populations (Nr_-.(and N4) can be distinguished by their susceptibility to extraction from nuclei with 0.2 and 0.4 M KCl. Our studies show that these two forms represent discrete physiological entities, which differ in intact cells by: (a) their rates of formation under conditions which are not dependent on the initial steroidreceptor interaction, the N4 fraction being formed more rapidly; (b) their rates of dissociation following a reduction in hormone concentration which reduces the N4 fraction more rapidly; (c) their response to a cold chase of unlabeled steroid which affects the N, fraction only very slowly; (d) their distribution within nuclei when thymocytes are exposed to glucocorticoids of different biological potency, the N4 fraction predominating with thi most potent steroids. Recently, Mueller et al. [22] have challenged the validity of KC1 extraction for distinguishing classes of nuclear estrogen receptors. They show that reextraction of nuclei from rats given a single injection of one concentration of C3H]-estradiol leads to continual extraction of tritium. In their studies they used a low ratio of extraction solution to nuclei (9: I, v/v), which probably accounts for the incomplete initial extractions which they observed. We have used a high ratio (25: 1, v/v), and as shown in Fig. 2, no further extraction of either the N2 or N, fractions occurs when they are exposed respectively, to a second extraction with 0.2 or 0.4M KCI, indicating that one extraction with either 0.2 or 0.4M KC! gives complete extraction for that KC1 concentration. Furthermore, since the fraction resistant to 0.2 M KC1 can be further extracted by 0.4M KC1 until it reaches the level of the N, fraction, we conclude that the N4 and N2_-4 fractions represent different forms of nuclear glucoeorticoid receptor complexes. Recent evidence obtained by using Triton X-100 to solubilize the outer nuclear membrane from thymocyte nuclei indicates that the tightly bound N4 fraction resides principally within the nuclei whereas the
111
N 24 fraction appears to be localized on the outer nuclear membrane (Cidlowski, in preparation). Recently, Milvihill and Paimiter[23] demonstrated an excellent correlation between nuclear progesterone binding with production of ovalbumin mRNA in the chick oviduct. Their nuclear preparations were treated with the 0.25% Triton X-100, and probably were free of the outer nuclear membrane which we suggest may contain the N,_, fraction. This observation suggests that under physiological conditions in which cells are normally exposed to low steroid hormone concentration (1O-9-1O-s M) occupation of N, sites may predominate. Several studies have shown that glucocorticoid binding in whole cells is rapidly reduced folIowing the addition of a cold chase of unlabeled hormone [4, 181. With thymus cells, the levels of nuclear binding were found, rather surprisingly, to decrease prior to reduction in cytopl~mi~ receptor level [4]. This result has been interpreted to suggest direct interaction of free steroid with nuclear-bound receptors. As the data in Figs 5 and 6 indicate, the N,_, and N4 fractions show remarkably different kinetic behavior following a chase of unlabeled dexamethasone, the N4 fraction being considerably more resistant than the N2_-Q.This observation is consistent with the steroid being bound more tightly in the N, than the N,_, fraction. Alternatively it could indicate that addition of hormone causes a transformation of NZ_-4into N4 complexes. So far, however, all our attempts to measure directly such a precursor-product relationship between N2_., and N, have been negative. In contrast to the results which indicate that N4 is the most strongly bound fraction, our observation that the N, fraction decreases more rapidly than the N,_, fraction after reduction in hormone con~ntration is striking. We have no good explanation for this paradox. Clark and Peck [S], studying estrogen receptors in rat uterus, have shown that occupation of nuclear sites resistant to extraction with 0.4 M KC1 correlates well with stimulation of long-term uterine growth. Our data, particularly those in Table 1 indicating that the most potent glucocorticoids have the largest N, fraction, also suggest that the N4 fraction may be important for ghrcocorticoid responses in thymocytes. The lesser physiological importance of the NZ_-4 fraction is suggested by the similar percent occupation in this fraction by cortisol, dexamethasone. and triamcinolone acetonide (Table l), indicating that this fraction does not discriminate between steroids with widely differing glucocorticoid activities. In addition, the anti-glucocorticoids cortexolone progesterone both displace the NZ_-4fraction almost as effectively as dexamethasone in cold chase experiments. Implicit in the existence of several nuclear fractions are at least two possibilities which are not mutually exclusive. One is that the primary difference between the fractions lies in the nature of the nuclear binding sites, and that there are several distinct subpopulations of these sites. This possibility is supported by
112
JOHN A. CI~LOWSKIand ALLANMUNCK
the preliminary results with Triton X-extracted nuclei mentioned above. The second possibility is that the different nuclear fractions are formed by distinct types of cytoplasmic complexes. Some preliminary results (Munck and Foley, unpublished) suggest that the cytoplasm of rat thymocytes at 37°C may contain
I I. Defer N., Dastugue B. and Kruh J.: Rat liver chromatin non-histone proteins and glucocorticoid binding. , 2, Biochimie 56 (1974) 1549-1557. Puca G. A., Nola E., Hibner U., Cicala G. and Sica V.: Interaction of the estradiol receptor from calf uterus with its nuclear acceptor sites. J. biol. Chem. 250 (1975)
more
L. J. and Ruh T. S.: Antiestrogen action: 13. Baudenistel Differential nuclear retention and extractability of the estrogen receptor. Steroids 28 (1976) 223237. II. Jackson V. and Chalkley R.: The binding of estradial-17/I to the bovine endometrial nuclear membrane, J. biol. Chem. 249 (1974) 1615-1626. 15. Clark J. H., Anderson J. N. and Peck E. J. Jr: Nuclear receptor-estrogen complexes of rat uteri: Concentration and time response parameters. In ReceptorsJor Reproductiue Hormones (Edited by B. W. O’Malley and A. R. Means). Plenum Press, New York (1973) pp. 15-59, Vol. 36. 16. Baxter J. D., Rousseau C. G., Benson M. C., Garcea R. C.. Ito J. and Tomkins G. M.: Role of DNA and specific cytoplasmic receptors in a glucocorticoid action. Proc. natn. Acad. Sci., U.S.A. 69 (1972) l892- 1896. 17. Rousseau G. G., Higgins S. J., Baxter J. D., Gelfand D. and Tomkins G. M.: Binding of glucocorticoid receptors to DNA. J. biol. Chem. 250 (1975) 6015-6021. J. L., Wong M. D., Ishii D. N. and 18. Middlebrook Aronow L.: Subcellular distribution of glucocorticoid receptors in mouse fibroblast. Biochemistry 14 (1975) 180-186. A. and Wira C.: Methods for assessing 19. Munck hormone-receptor kinetics with cells in suspension: Receptor-bound and nonspecifically bound hormone; cytoplasmic-nuclear translocation. In Methods in Enzymologp (Edited by B. W. O’Malley and J. Hardman). Acadmic Press, New York (1975) PP. 25>266. Vol. 36. properties 20. Bell P. A. and Munck A.:’ Sterbjd-binding and stabilization of cytoplasmic glucocorticoid receptors from rat thymus cells. Biochem. .I. 136 (1973) 977107. 21. Mosher K. M., Young D. A. and Munck A.: Evidence for irreversible, actinomycin D-sensitive, and temperature-sensitive steps following binding of cortisol to glucocorticoid receptors and preceding effects on glucose metabolism in rat thymus cells. .f. biol. Chem. 246 (I 97 1) 654659. 22. Mueller R. E., Traish A. M. and Wotiz H. H.: Interaction of receptor-estrogen complex with uterine nuclei. J. biol. Chem. 252 (1977) 8206821 I. R. D.: Relationship of 23. Mulvihill E. R. and Palmiter nuclear estrogen receptor levels to induction of ovalbumin and conalbumin mRNA in chick oviduct. J. hiol. Chem. 252 (1977) 2060-2068.
than
complex. lation
one form
of “activated”
We are currently
of these
forms
trying
hormone to establish
to nuclear-bound
receptor the re-
complexes.
Ack,lowledgements-Supported by Research Grants AM03535, CA 17323, AM 20892 and Fellowship AM-05081, AM-20892
from the U.S. Public
Health
Service.
REFERENCES T.: Specific and nonI. Munck A. and Brinck-Johnsen specific physicochemical interactions of glucocorticoids and related steroids with rat thymus irl oirro. J. biol. Chum. 243 (1968) 55565565. 2. Wira C. R. and Munck A.: Glucocorticoid&eceptor complexes in rat thymus cells. Cytoplasmic-Nuclear Transformations. J. biol. Chem. 249 (1974) 5328-5336. receptors and 3. Munck A. and Leung K.: Glucocorticoid mechanisms of action. In Receptors and Mechanism of Action of Steroid Hormones (Edited by J. R. Pasqualini). Marcel Dekker, New York (1976) pp. 31 l-397, Part II. 4. Munck A. and Foley R.: Kinetics of glucocorticoidreceptor complexes in rat thymus cells. J. steroid Biothem. 7 (1976) II 17-l 122. K. R. and Alberts B. M.: Steroid receptors: 5. Yamamoto Elements for modulation of eukaryotic transcription. An. Rev. Biochem. 45 (1976) 721-744. 6. Spelsberg T. C., Pikler G. M. and Webster R. A.: Progesterone binding to hen oviduct genome: specific versus nonspecific binding. Science 194 (I 976) 197-l 98. 7. Spelsberg T. C., Steggles A. W. and O’Malley B. W.: Progesterone-binding components of chick oviduct III: “Chromatin Acceptor Sites”. J. hiol. Chern. 246 (1974) 41884197. of 8. Clark J. H. and Peck E. J. Jr: Nuclear retention receptor-oestrogen complex and nuclear acceptor sites. Nafure 260 (1976) 635-637. L., Chiu J, Tsai Y. and Hnilica 9. Klyzsehko-Stefanowicz L.: Acceptor proteins in rat androgenic tissue chromatin. Proc. narn. Acad. Sci.. U.S.A. 73 (1976) 19541958. IO. Simons S. S. Jr. Martinez H. M., Garcea R. L.. Baxter J. D. and Tomkins G. M.: Interactions of glucocorticoid receptor-steroid complexes with acceptor sites. J. hiol. Chem. 251 (1976) 334343.
6452-6459.
MULTIPLE FORMS OF NUCLEAR BINDING OF GLUCOCORTICOID-RECEPTOR COMPLEXES IN RAT THYMOCYTES JOHN A. CIDLOWSKI* and ALLAN MUNCK Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire, U.S.A. (Recriurd 18 April 1979) SUMMARY Exposure of isolated rat thymocytes to glucocorticoids at 37°C leads to localization of 50~-80~ of the o$ular glucocorticoid-receptors on or within the nucleus. In this study, we have used differential salt extraction of nuclei from cells incubated with glucocorticoids under various conditions to further characterize the nature of the nuclear glucocorticoid-receptor interaction. Of the dexamethasone initially bound to nuclei, 70-75% is resistant to 0.2 M KC1 salt extraction and 2$_30% is resistant to 0.4 M KC1 salt extraction. These two resistant fractions are designated N, and N, respectively. The N, fraction contains N4 since Nz can be further extracted with 0.4 M KCl, whereas N4 is resistant to further extraction with either 0.2 or 0.4 M KCl. These data suggest that Na and the difference N2_.&= N, - N& (i.e. that fraction which is resistant to 0.2 M but not to 0.4 M KC?) represent distinct forms of nuclear dexamethasone-receptor interaction. In intact cells at 37°C both N2_., and N, are formed at all the steroid concentrations, and neither fraction shows evidence of saturation. The two fractions differ, however, in the following ways: (a) N4 is formed prior to N,_, under conditions where the rate of formation of the cytoplasmic dexamethasone-receptor complex is not limiting; (b) the addition of a cold chase of unlabeled dexamethasone to cells pre-exposed to [‘HI-dexamethasone decreases N,_, after 60 min by about 70”/, but N, by only 10-15%; (c) even at concentrations of unlabeled dexamethasone which do not decrease total cellular binding a cold chase causes a sharp reduction in N,_, but not in N,; (d) N, decreases more rapidly than N,_, following a decrease in hormone concentration by dilution; (e) the magnitude of the dilution determines the extent of N4 reduction, and little or no change occurs over the 35-90 fold range; (f) cold chases with the anti-glucocorticoids cortexolone and progesterone significantly decrease N2_&but had little effect on N,. The distribution of Rluc~orticoid-re~ptor complexes in the N,_, and N, fractions depends on the steroid which is bound to the receptor. Triamcinoione acetonide,which has similar in r&o biological potency to dexamethasone, has a similar N4 fraction (29%) of nuclear receptor-bound steroid but a greater N,_, fraction (60%). Cortisol, which is 10% as active as dexamethasone, has an N4 fraction of only 13% and an Ns_.+ fraction of 41%. These data indicate that glucocorticoids form physiologicaily distinct classes of nuclear acceptor-receptor complexes.
INTRODUC’HON
Rat thymic lymphocytes respond to physiological levels of glucocorticoids with rapid alterations in cellular metabolism and contain receptors with high affinity and specificity for both natural and synthetic glucocorticoids [l-4]. Hormone-receptor complexes formed in the cytoplasm are rapidly translocated to the nucleus at 37°C and under steady-state conditions most of the receptor-bound steroid is in the nucleus [Z, 43. Since most of the known metabolic responses appear to require RNA synthesis [3], the nucleus seems a likely target for the initiation of responses to glucocorticoids. Recently, considerable effort has been directed at determining the nature of nuclear hormone-receptor binding sites for all classes * Present Address: Department of Biochemistry, University of Vermont. Dexamethasone = (9 a-fluoro-I 1& 17, 2l-trihydroxy-~6ff methylpregna-1,4,-diene,-3,20-dione): triamcinolone acetonide = (9 a-fluoro-1 I/I’, I&X, 17x, 21, tetrahydroxy pregna1,4-diene-3, 20 dione-16, 17-acetonide); cortisol = (I Is, 17a, 21 trihydroxy-4 pregnene-3, 20 dione). 5.8.
13/2--n
of steroid-receptor complexes [ 5- 143. DNA [S, lo], nuclear acidic proteins [6,7,9, 1l] and nuclear membranes Cl43 all have been suggested as possible sites. The approach used in most of these investigations has been to expose cells in uiuo or in culture to a single con~ntration of radioactive hormone, then to isolate nuclei and chromatin, and determine the hormone bound to chromatin subfractions. Studying the actions of estradiol on rat uterus, Clark et al.[lS] have taken an alternate approach using differentia1 salt extractions. They have found that long-term retention of hormone in a fraction of nucleic that is unextractable by 0.4 M KC1 correlates well with uterine growth. Similar high-salt residual nuclear fractions were originally observed with glucocorticoids in hepatoma cells [ 16,171 and subsequently in cultured fibroblasts [18], the latter studies indicating that the salt-residual form of nuclear glucocorticoid receptors increases with energy deprivation. In the present studies we have taken a physiological approach to the characterization of different nuclear forms of glucocorticoids-receptor complexes in 105
JOHN
106
A. CIDL~WSKI
thymus cells, measuring the distribution and kinetic behavior of salt-extractable and unextractable nuclear complexes in intact cells exposed to various conditions which mimic those to which cells are exposed in the whole organism. We present evidence that thymocytes contain at least two distinguishable classes of nuclear glucocorticoid-receptor complexes which associate in unique manners with nuclei. The distribution of these classes of glucocorticoid receptors within whole cells is dependent on steroid concentration, length of time of hormone exposure and biological potency of the steroid molecule. METHODS General
Thymus tissue was obtained from male SpragueDawley rats that had been adrenalectomized 6-8 days prior to.sacrifice and mainlined on 0.9% NaCl and rat chow. Thymus cell suspensions were prepared in Krebs-Ringer Bicarbonate Buffer with lOmM glucose (KRBG) equilibrated with 95% oxygen; 5% CO1 as described previously [I]. The cytocrit (packed cells per ml. of cell suspension) of each preparation was adjusted to 0.2-0.3. Unlabeled dexamethasone, cortexolone and progesterone, purchased from Steraloids, were prepared in concentrated stock solutions ( 10e4 M) in KRBG, and were added to cell suspensions to give the concentrations indicated. [6,7-‘HIDexamethasone (33.0 Ci/mmol), [ 1,23H]-cortisol (45.8 Ci/mmol) and ~4,73H]-tri~cinolone acetonide (33.7 Ci/mmol) were obtained from New England Nuclear. Aliquots of solutions of radioactive steroids dissolved in 9w; benzene, 10% ethanol were placed in the incubation vials, and the organic solvents evaporated to dryness prior to the addition of cell suspensions. The concentrations of steroids given in the results are the total average concentrations. The effective concentrations of free steroid to which the cells are exposed are lower because a substantial fraction of the steroid is bound, most of it nonspecifically [I]. Approximate factors by which the total concentrations should be multiplied to give the free concentrations, taking into account the cytocrit of the cell sus~nsion [i] are: dexamethasone, 0.4; cortexolone, 0.6; progesterone, 0.13. fncubatiun of thymus ceils with steroid and measurement ofcytoplasmic and nuclear receptor binding Suspensions of thymus cells were incubated with tritiated steroids at l-2 x lo-* M in the presence or absence of unlabeled dexamethasone 2 x 10m6M. Receptor-bound tritiated steroid was determined as the difference between values obtained without and with unlabeled hormone. The cell suspensions (generally 300-500~1) were incubated in 1 dram screw-cap glass vials or lOm1 Erlenmeyer flasks which were flushed with 95% 0,:5x COz. The vials were shaken at 20 cycles per minute in a water bath maintained 37°C. Cytoplasmic binding was measured by placing
and ALLAN MUNCK
a 20~1 aliquot of the thymus cell suspension into lOO$ of dextran-coated charcoal in 1.5 mM MgCl, at O”C, vortexing the mixture, leaving it for 15 min at 3°C and sedimenting the charcoal and cellular debris at 15,OOOgfor 2min [19]. Samples of the supernatant were then removed for assessment of radioactivity. Except where a different procedure is described in the figure caption, nuclear binding was measured by placing a 20~1 aliquot of thymus cell suspension into 15 ml polyethylene tubes containing lOm1 of 1.5 mM MgCf, at O”C, incubating this suspension for 15 min at 3”C, sedimenting the nuclei by centrifugation at 3000 g for 5 min. The supernatants were decanted and the tips of each tube containing the nuclear pellet cut off and placed into a liquid ~intillation vial[i9]. Both cytoplasmic and nuclear samples were counted for radioactivity in 5.0ml of Brays solution using a Packard Tri-Carb liquid scintillation counter with efficiency of 25% for tritium. Salt extraction procedures Nuclear pellets from the cells in 20~1 of a suspension that had been incubated with tritiated steroid alone or tritiated steroid plus unlabeled dexamethasone were prepared by breaking the cells in lOm1 of 1.5mM MgCll and centrifuging as described above. These nuclei were then extracted with 500$ of ice cold 1.5 mM MgCl, (control), 0.1 M KCI, 0.2 M KCl, 0.3 M KCl, 0.4 M KC1 or 100% ethanol in the same 15 ml polyethylene tube used in the preparation of the nuclei. After addition of each solution, the pellets were vortexed vigorously for 10s and placed on a Thomas rotating apparatus at speed setting No. 7-8 (60 cycles/min) for 30min at 3°C; under these conditions the pH of the extraction solutions was approximately seven. The pellets were then immediately centrifuged at 3OOOg for 5 min, and lOO/*l of the supernatants taken for measurement of radioactivity. The remaining solution and pellet were diluted by adding lOm1 of 1.5 mM MgCl, at 0°C followed immediately by centrifugation at 3000g for 5 min, the supernatants decanted and the tips of each tube containing the residual nuclear pellet cut off and placed in liquid ~ntil~tion viafs for counting. These procedures give a measure of both extracted and residual nuclear-bound radioactivity. The conditions of these extractions yielded maximal extraction and were independent of the amount of nuclei used, up to nuclei from 100 /.d of cell suspension. All results are given as the difference between the bound cpm from the incubations without and with added unlabeled dexamethasone. RESULTS
Nuclear distribution of glucocorticoid receptors in rat tjymocytes Figure 1 shows a salt-extraction pattern of receptor-bound dexamethasone from thymocyte nuclei. Control nuclei were extracted with 1.5 mM
. L
Heterogeneity of nuclear glucocorticoid-receptor
,cpm
Cpm
E300 51 .z Q E s 200 8 -z 2 m
KCI (M)
Fig. I. Salt extraction pattern of dexamethasone-receptor from nuclei of cells exposed to [“HI-dexamethasone. Suspensions of thymus cells were incubated with [“HI-dexamethasone with or without dexamethasone in Erlenmeyer flasks for 30min at 37°C as described in Methods. Nuclei were prepared and extracted as outlined in the Methods. The data shown represent residual nuclear radioactivity values obtained from a typical experiment. Extractions were carried out in quadruplicate. Results are the means f standard errors obtained after subtracting the non-saturable counts in the presence of unlabeled dexamethasone.
MgClz in order to assess steroid dissociation during the 3°C extraction procedure. Less than 5% of the steroid dissociated from the receptor under these conditions. Of the dexamethasone receptor initially bound, about looO/, is resistant to 0.1 M KCI, 7&75x is resistant to 0.2 M KC1 and 25% is resistant to 0.4 M KCI. Higher salt concentrations (0.5 and 0.6 M KCI) did not lead to any further extraction and caused the nuclei to form a jelly-like mass. For subsequent results, receptor binding resistant to extraction by a particular KC1 concentration is designated as follows: N, is the fraction resistant to extraction by 0.1 M KCl, N, is the fraction resistant to extraction with 0.2 M KCl, etc. When the extracts of nuclei treated with 0.4 M KC1 were exposed to dextran-coated charcoal approximately 50% df the total extracted radioactivity was not absorbed and so was presumably in the steroid-receptor complex form. All of the nuclearbound dexamethasone was extracted following treatment of the nuclei with lOOo/,ethanol. In each experiment we have also measured the amount of radioactivity extracted; we find excellent correlation between total initial binding, extracted steroidreceptor complex and the resistant fractions. For simplicity, we show only the resistant fraction. Similar salt extraction patterns of nuclear glucocorticoid receptor complex have been observed for hepatoma cells [ 163, and fibroblasts [ 183. The data shown in Fig. 2 demonstrate that only extraction solutions of higher ionic strength are effective in removing nuclear-bound dexamethasone resistant to previous salt extraction. A second extraction with 0.1 M or 0.2 M KC1 does not decrease the N2 or N4 fractions while second extraction with 0.3 M or 0.4M KCl, however, decreases the N, fraction down to the N4 level. These studies suggest three points: (a) the first extractions are maximally effective at each
107
interactions
1
q
of Nq
b 100 h J B
OF&fore t IO.1 IO.2 IO.3 Reextraction Clont roi KCI (M)
f0.4,IEtO
.
id
Fig. 2. Re-extraction of N2 and N4 fractions of nuclearbound dexamethasone-receptor. Suspensions of thymus cells were incubated as for the experiments in Fig. I. Nuclei were prepared and extracted with either 0.2 M KCI or 0.4 M KC1 as described in Methods. The pellets were then resuspended in 10 ml 1.5 mM MgClz at 0°C and the suspensions immediately centrifuged at 3000 g for 5 min. The supernatants were discarded and some of the pellets containing N2 and N4 fractions were used for measuring radioactivity (values designated before) and the rest were extracted a second time with 0.1 M KCI, 0.2 M KCI, 0.3 M KCl, 0.4M KC1 or lOOo/,ethanol. The values shown are mean of quadruplicate measurements. Error on these determinations was not greater than 5% on different samples of nuclei.
salt concentration; (b) the N, fraction is contained in the N, fraction, or in other words, the fraction resistant to 0.4 M KCI is also resistant to 0.2 M KCl, but not vice-versa; (c) the salt extraction procedure defines two more-or-less discrete fractions, N, which is resistant to 0.4 M KCl, and the difference N,_, = N2 - N4 which is resistant to 0.2 M KC1 but not to 0.4M KCl. Our subsequent results are expressed in terms of Nz. N*, the calculated N2_-4and the amount bound to unextracted whole nuclei. The data in Fig. 3 shows the dependence of these fractions on [‘HI-dexamethasone concentration. N,, N, and N,_, are present at every concentration in roughly the same proportions (except at the lowest concentrations, as noted below). Half maximal values occur at about 10 nM, the K, for binding of dexamethasone to the receptors, suggesting that the nuclear sites do not become saturated. There is, however, a slight indication of preferential occupation of the N4 sites at low hormone concentrations, below 10nM. We have obtained this result consistently in several experiments. Figure 4 shows the time-course of occupation of the N2_4 and N4 fractions after dexamethasone is added to cells. The N4 fraction increases rapidly and reaches a plateau by 3 min, then continues to increase slowly. The N, and NzA fractions increase for about 15 min before leveling out. Under the conditions of the experiment in Fig. 4, the overall rate-limiting step appears to be formation of cytoplasmic steroid-receptor complex [ 1,4]. A sep-
108
JOHN A CIDLOWSKI~II~ ALLAN
OL Dexamethasone
Concentration (nM)
Fig. 3. Influence of [3H]-dexamethasone concentration on the distribution of the nuclear-bound dexamethasone fractions. Suspensions of thymus cells were incubated with various concentrations of C3H]-dexamethasone with or without unlabeled dexamethasone for 30 min at 37°C. Nuclei from 20~1 of the cell suspension were prepared and either not extracted (whole nuclei) or extracted with 0.2 M and 0.4 M KC1 for determination of N, and N, fractions
MUNCK
carried out this experiment twice, with the same result. We have recently found that the addition of a “chase” of unlabeled dexamethasonone to cells previously equilibrated with [3H]-dexamethasone leads to a decrease in nuclear binding of C3H]-dexamethasone that is considerably faster than the decrease in cytoplasmic binding [4]. The experiments in Fig. 6 examine the kinetics of a cold chase of unlabeled dexamethasone on the rate of loss of the N, and N,-, fractions. Total nuclear dexamethasone decreases rapidly with 60”/, of the initially bound hormone lost after 60 min. During this time, NzP4 decreases in proportion to the total nuclear binding, whereas N, is reduced only marginally. In Fig. 7 we show the magnitudes of the various nuclear fractions 1 h after chase at 37°C with several concentrations of dexamethasone. Here the N,_, fraction again is displaced to a much greater degree than the N4 fraction. A particularly striking observation is
as described m Methods. Values represent the means of at least four separate extractions on different aliquots of cells. N 2m4were calculated as N, - N,.
2000
.
1500
. I'
arate experiment was therefore designed to measure formation of N, and N2_-4 under conditions where the rate of activation of steroid-receptor complex is ratelimiting [2,4]. Cytoplasmic steroid-receptor complex was formed by incubation of whole cells with steroid at 0°C for about 3 h. Using a procedure developed previously [2], the cells were warmed rapidly and kept at that temperature for the number of seconds indicated in Fig. 5. The data shown in Fig. 5 show that the N4 fraction begins to be formed immediately on warming, but the N,_, fraction is formed very slightly if at all. This experiment indicates that the N, fraction is formed prior to the N,_, fraction. We have
:
l:
500-
Dexomethasone
Fig. 4. Time-course of formation of N,, N4 and N,_, fractions after addition of dexamethasone to thymus cells at 37°C. Suspensions of thymus cells were incubated with [3H]-dexamethasone with or without unlabeled dexamethasone at 37°C for the time period indicated. The reactions were stopped by pipetting 20~1 of cells into IOml of ice cold I .5 mM MgCl,, followed by preparation of nuclei and measurement of the N2 and N4 fractions as described in Methods. Each value is the mean of four separate determinations. N,-, = N, - N,.
lW
"'
NUCLEI
0
20 SECONDS
After
.
lti
WHOLE
.
%
0
Mknutes
.
. . .
40 WARMED
80
60 TO
37”
C
Fig. 5. Time-course of formation of N,, N, and N,-, fractions after cells with preformed dexamethasoneereceptor complexes are warmed to 37°C. Suspensions of thymus cells were incubated with [‘HI-dexamethasone with or without unlabeled dexamethasone for 18c-240 min at 0°C. Triplicate 20 ~1 aliquots were removed and added to 200 ~1 KRBG at 37°C in 15 ml conical polyethylene centrifuge tubes with the tips in a 37°C water bath. For the O-s time points the cells were placed in 200~1 KRBG at 0°C. After the indicated number of seconds the cells were simultaneously cooled and disrupted by addition of 1Oml 1.5 mM MgCI, at 0°C. The resulting suspensions were centrifuged to obtain nuclear pellets. One of these was used to obtain counts for whole nuclei, and the other two were extracted to determine the N, and N, fractions, and N 2-4 = N, - N,. Each point is the result from one pellet, corrected for saturable binding.
Heterogeneity
of nuclear
glucocorticoid-receptor
109
interactions
7
Whole
Nuclel
After
Dilution
9) 100 i
o- $S&.-:;
IO Minutes Minutes
After Dexamethasone Chase
Fig. 6. Time-course of loss of nuclear-bound [‘HI-dexamethasone fractions following a cold chase of unlabeled dexamethasone. Suspensions of thymus cells were incubated with [3H]-dexamethasone, with or without unlabeled steroid for 30min at 37°C. Twenty-PI aliquots of cell suspension were removed for measurement of the whole nuclear binding and the N, and N, fractions. Unlabeled dexamethasone was subsequently added to each group to give a final concentration of 2 PM, and the incubations continued. At the intervals indicated, samples of cell suspension were removed for measurement of total nuclear binding, and the N4 and N2 fractions. The values are mean values of four separate extractions from different aliquots of cells. N,_, = N, - N.,.
that at low concentrations of unlabeled dexamethasone (3 x lo-’ M) which hardly affect whole nuclear dexamethasone or N,, there are significant decreases in the N,_, fraction. The next experiments (Fig. 8) examine the influence of removal of the [3H]-dexamethasone on the timecourse of loss of total nuclear dexamethasone binding and of the N,_, and N, fractions. Thymus cells pre-
Fig. 7. The influence of chases with unlabeled dexamethasone at various concentrations on nuclear binding after 1 h. Ten groups of cell suspensions, 200 ~1 each, were incubated with [‘HI-dexamethasone with or without dexamethasone for 30 min at 37°C. Unlabeled dexamethasone in 5 ~1 ahquots at concentrations from 10e4 M to 6.25 x 10e6 M, was added to each group to give the final unlabeled dexamethasone concentrations indicated. The incubations were continued at 37°C for an additional 60min. Total nuclear dexamethasone-receptor binding and the N, and N, fraction were measured in nuclei from four separate 20~1 aliquots of cells. Each point represents the mean value for residual binding. N,_, = N, - N,.
20
3(
Fig. 8. The influence of removal of [3H]-dexamethasone on the time-course of loss of nuclear dexamethasonereceptor. Two l.Oml aliquots of thymus cell suspensions were equilibrated with C3H]-dexamethasone with or without unlabeled dexamethasone for 30 min at 37°C and 20 ~1 aliquots were removed for measurement of total nuclear dexamethasone-receptor and the N, and N, fractions. Twenty ~1 aliquots of cell suspension were placed into 2.0ml of KRBG at 37°C and allowed to incubate for the periods of time indicated. The dilution reaction was stopped at each time interval by adding 8.0ml of ice cold 1.5 mM MgCI,, followed by immediate centrifugation at 3000 g for 5 min. The supernatant of each tube was then decanted, nuclei were prepared by resuspending the pellet in 1.5 mM MgCI,, etc, and total nuclear dexamethasonee receptor and N, and N, fractions were measured. N 2-4 = N, - N,.
viously incubated with [3H]-dexamethasone with or without unlabeled hormone were diluted lOO-fold into KRBG for the intervals of time indicated. Total nuclear binding and the N, fraction rapidly decrease after dilution with little measurable N4 remaining in the nuclei after 30min. Surprisingly in view of the results from the chase experiments, there is little if any decrease in the N,_, fraction. These data show that the N, fraction is more sensitive than the N,_, fraction to reductions in C3H]-dexamethasone con-
Dllutton
Fig. 9. The influence of the magnitude of dilution on the decrease in nuclear bound fraction. Cell suspensions were incubated with [3H]-dexamethasone with or without unlabeled dexamethasone as described in Methods. Twenty ~1 aliquots of cell suspension were then diluted into different volumes of KRBG at 37°C with the dilution ratios indicated. All groups of cells were then allowed to incubate for an additional 30 min at 37°C. The dilution reactions were stopped by the addition of 1.5 mM MgCI, at 0°C and the cell pellets were processed as described in Fig. 8. Values are the mean of determinations from three to four different aliquots of cells.
110
JOHN A.
CIDLOWSKIand ALLANMUNCK
Table I. Distribution of nuclear bound-steroid receptor complexes for cortisol, dexamethasone and triamcinolone acetonide
Steroid Dexamethasone Triamcinolone Cortisol
Nuclear bound steroid Total (moU 0 2.6 x IO-l4 5.2 x IO-“’ 0.5 x lo-l4
80 90 54
2 29 29 14
%ii 50 61 40
Thymus cells were incubated with 2.0 x IO-* M of each steroid with or without 4 x 10e6 M unlabeled dexamethasone for 30 min at 37°C. Total nuclear binding, N, binding and N, binding was then measured in nuclei from 20~1 aliquots of cell suspensions. Residual binding values are the mean of two separate experiments. N,_, = N, - Nq. centration. Similar results have been obtained with [3H]-cortisol and [3H]-trimcinolone acetonide (Cid-
lowski and Munck, unpublished). Figure 9 shows how the magnitude of the reduction in whole nuclear binding and the NzA and N* fractions depends on the extent of reduction of hormone concentration by dilution. Dilution of cells by lOOfold for 30min at 37°C reduces the N4 fraction by 95% and the N,_, by 55%. Dilutions of lesser magnitude cause smaller total reduction in nuclear binding but always lead to a greater loss of N4 than N2_& As shown in Table 1, following exposure of thymocytes to equal molar concentrations of dexamethasone, cortisol and triamcinolone acetonide, unequal amounts of steroid are associated in the nuclei. These values correlate well with the affinities of these
Minutes
After Chase
Cortexolone
Fig. 10. The influence of cold chase of unlabeled cortexolone on the time-course of loss of nuclear dexamethasonereceptor. One ml aliquots of thymus cell suspensions were equilibrated with [3H]-dexamethasone with or without unlabeled dexamethasone for 30 min at 37°C and 20~1 samples of cell suspension removed for measurement of each nuclear binding fraction. To the remaining cell suspension, lOpI of 10m4M unlabeled cortexolone in KRBG was added to give a final concentration of 10e6 M, and the incubation continued. Total nuclear binding, N, and N4 nuclear binding were measured in nuclei from 20~1 aliquots of cell suspension removed at the times indicated. The values shown are then mean f standard error from determinations of four separate samples at each time point. N,_, = N, - N.,.
00 Minutes
15
30
After Chase
45
6C
Progesterone
Fig. Il. The influence of a cold chase of unlabeled progesterone on the time-course of loss of nuclear dexamethasone-receptor. One ml aliquots of cell suspensions were equilibrated with [“HI-dexamethasone as described and total nuclear binding and the N, and N4 fractions measured in nuclei from 20~1 aliquots. To the remaining cell suspension 40 ~1 of 2.0 x IO-’ M unlabeled progesterone prepared in KRBG was added to give a final concentration of 8 x IO-‘M and the incubation of cell suspension continued. After the indicated times, 2O/.d aliquots of cells were removed for assessment of nuclear binding. Each value represents the mean f standard error of quadruplicate determinations. N2_4 = N2 - N4..
for the cytoplasmic glucocorticoid receptor [3]. As observed earlier (Fig. l), nuclear dexamethasone-receptor complexes are relatively resistant to 0.2M KC1 extraction (N2), as are those for nuclear triamcinolone acetonide-receptor complexes. However, only 54% of the initially bound cortisol-receptor complexes are resistant to 0.2 M KCl. Cortisol, dexamethasone and triamcinolone acetonide receptor complexes all have similar N2_4 fractions (4&61x) and dexamethasone and triamcinolone acetonide, two steroids with equal in vitro biological activity (4), have similar N4 binding (29%). The N, fraction for the weaker glucocorticoid, cortisol, is about half that of either synthetic compound. Thus, the natural and synthetic glucocorticoids form different types of nuclear complexes. Figures 10 and 11 respectively, show the results on nuclear-bound [3H]-dexamethasone of chases with cortexolone and progesterone. Both these steroids are known to have anti-glucocorticoid activity [3]. Both of these steroids decrease total nuclear binding of [‘HI-dexamethasone after 60min. During the first 15 min, before whole nuclear binding has decreased, cortexolone initiates a rapid reduction in the NZ_, fraction with little alteration in the N4 fraction. The chase with progesterone is less effective than with cortexolone, probably due to the lower free progesterone concentration used (see Methods). The data in Fig. I1 show, however, that unlabeled progesterone is also capable of causing significant reductions in whole nuclear binding and in the NZ_., fraction, but fails to significantly alter the N4 fraction. steroids
Heterogeneity of nuclear glucocorticoid-receptor interactions DISCUSSION
Several investigators studying the mechanism of steroid hormone action have suggested that heterogeneity exists in the nature of steroid-receptor complexes [8,1 l-143. These suggestions were prompted by observations that maximal physiological responses could be obtained by exposure of cells to concentrations of steroid which would not occupy all available cytoplasmic steroid receptors, and that subpopulations of nuclear steroid-receptor complexes show differential susceptibility to extraction with KC1 [S, 151. Using isolated thymocytes, a system with which one can conveniently obtain nuclei from cells treated with a variety of hormonal conditions, we have sought to probe the interactions of glucoeorticoidreceptor complexes with nuclei. Our studies demonstrate (Figs 1 and 2) the presence of at least two different populations of glucocorticoid-receptor complexes within or on thymocyte nuclei. These populations (Nr_-.(and N4) can be distinguished by their susceptibility to extraction from nuclei with 0.2 and 0.4 M KCl. Our studies show that these two forms represent discrete physiological entities, which differ in intact cells by: (a) their rates of formation under conditions which are not dependent on the initial steroidreceptor interaction, the N4 fraction being formed more rapidly; (b) their rates of dissociation following a reduction in hormone concentration which reduces the N4 fraction more rapidly; (c) their response to a cold chase of unlabeled steroid which affects the N, fraction only very slowly; (d) their distribution within nuclei when thymocytes are exposed to glucocorticoids of different biological potency, the N4 fraction predominating with thi most potent steroids. Recently, Mueller et al. [22] have challenged the validity of KC1 extraction for distinguishing classes of nuclear estrogen receptors. They show that reextraction of nuclei from rats given a single injection of one concentration of C3H]-estradiol leads to continual extraction of tritium. In their studies they used a low ratio of extraction solution to nuclei (9: I, v/v), which probably accounts for the incomplete initial extractions which they observed. We have used a high ratio (25: 1, v/v), and as shown in Fig. 2, no further extraction of either the N2 or N, fractions occurs when they are exposed respectively, to a second extraction with 0.2 or 0.4M KCI, indicating that one extraction with either 0.2 or 0.4M KC! gives complete extraction for that KC1 concentration. Furthermore, since the fraction resistant to 0.2 M KC1 can be further extracted by 0.4M KC1 until it reaches the level of the N, fraction, we conclude that the N4 and N2_-4 fractions represent different forms of nuclear glucoeorticoid receptor complexes. Recent evidence obtained by using Triton X-100 to solubilize the outer nuclear membrane from thymocyte nuclei indicates that the tightly bound N4 fraction resides principally within the nuclei whereas the
111
N 24 fraction appears to be localized on the outer nuclear membrane (Cidlowski, in preparation). Recently, Milvihill and Paimiter[23] demonstrated an excellent correlation between nuclear progesterone binding with production of ovalbumin mRNA in the chick oviduct. Their nuclear preparations were treated with the 0.25% Triton X-100, and probably were free of the outer nuclear membrane which we suggest may contain the N,_, fraction. This observation suggests that under physiological conditions in which cells are normally exposed to low steroid hormone concentration (1O-9-1O-s M) occupation of N, sites may predominate. Several studies have shown that glucocorticoid binding in whole cells is rapidly reduced folIowing the addition of a cold chase of unlabeled hormone [4, 181. With thymus cells, the levels of nuclear binding were found, rather surprisingly, to decrease prior to reduction in cytopl~mi~ receptor level [4]. This result has been interpreted to suggest direct interaction of free steroid with nuclear-bound receptors. As the data in Figs 5 and 6 indicate, the N,_, and N4 fractions show remarkably different kinetic behavior following a chase of unlabeled dexamethasone, the N4 fraction being considerably more resistant than the N2_-Q.This observation is consistent with the steroid being bound more tightly in the N, than the N,_, fraction. Alternatively it could indicate that addition of hormone causes a transformation of NZ_-4into N4 complexes. So far, however, all our attempts to measure directly such a precursor-product relationship between N2_., and N, have been negative. In contrast to the results which indicate that N4 is the most strongly bound fraction, our observation that the N, fraction decreases more rapidly than the N,_, fraction after reduction in hormone con~ntration is striking. We have no good explanation for this paradox. Clark and Peck [S], studying estrogen receptors in rat uterus, have shown that occupation of nuclear sites resistant to extraction with 0.4 M KC1 correlates well with stimulation of long-term uterine growth. Our data, particularly those in Table 1 indicating that the most potent glucocorticoids have the largest N, fraction, also suggest that the N4 fraction may be important for ghrcocorticoid responses in thymocytes. The lesser physiological importance of the NZ_-4 fraction is suggested by the similar percent occupation in this fraction by cortisol, dexamethasone. and triamcinolone acetonide (Table l), indicating that this fraction does not discriminate between steroids with widely differing glucocorticoid activities. In addition, the anti-glucocorticoids cortexolone progesterone both displace the NZ_-4fraction almost as effectively as dexamethasone in cold chase experiments. Implicit in the existence of several nuclear fractions are at least two possibilities which are not mutually exclusive. One is that the primary difference between the fractions lies in the nature of the nuclear binding sites, and that there are several distinct subpopulations of these sites. This possibility is supported by
112
JOHN A. CI~LOWSKIand ALLANMUNCK
the preliminary results with Triton X-extracted nuclei mentioned above. The second possibility is that the different nuclear fractions are formed by distinct types of cytoplasmic complexes. Some preliminary results (Munck and Foley, unpublished) suggest that the cytoplasm of rat thymocytes at 37°C may contain
I I. Defer N., Dastugue B. and Kruh J.: Rat liver chromatin non-histone proteins and glucocorticoid binding. , 2, Biochimie 56 (1974) 1549-1557. Puca G. A., Nola E., Hibner U., Cicala G. and Sica V.: Interaction of the estradiol receptor from calf uterus with its nuclear acceptor sites. J. biol. Chem. 250 (1975)
more
L. J. and Ruh T. S.: Antiestrogen action: 13. Baudenistel Differential nuclear retention and extractability of the estrogen receptor. Steroids 28 (1976) 223237. II. Jackson V. and Chalkley R.: The binding of estradial-17/I to the bovine endometrial nuclear membrane, J. biol. Chem. 249 (1974) 1615-1626. 15. Clark J. H., Anderson J. N. and Peck E. J. Jr: Nuclear receptor-estrogen complexes of rat uteri: Concentration and time response parameters. In ReceptorsJor Reproductiue Hormones (Edited by B. W. O’Malley and A. R. Means). Plenum Press, New York (1973) pp. 15-59, Vol. 36. 16. Baxter J. D., Rousseau C. G., Benson M. C., Garcea R. C.. Ito J. and Tomkins G. M.: Role of DNA and specific cytoplasmic receptors in a glucocorticoid action. Proc. natn. Acad. Sci., U.S.A. 69 (1972) l892- 1896. 17. Rousseau G. G., Higgins S. J., Baxter J. D., Gelfand D. and Tomkins G. M.: Binding of glucocorticoid receptors to DNA. J. biol. Chem. 250 (1975) 6015-6021. J. L., Wong M. D., Ishii D. N. and 18. Middlebrook Aronow L.: Subcellular distribution of glucocorticoid receptors in mouse fibroblast. Biochemistry 14 (1975) 180-186. A. and Wira C.: Methods for assessing 19. Munck hormone-receptor kinetics with cells in suspension: Receptor-bound and nonspecifically bound hormone; cytoplasmic-nuclear translocation. In Methods in Enzymologp (Edited by B. W. O’Malley and J. Hardman). Acadmic Press, New York (1975) PP. 25>266. Vol. 36. properties 20. Bell P. A. and Munck A.:’ Sterbjd-binding and stabilization of cytoplasmic glucocorticoid receptors from rat thymus cells. Biochem. .I. 136 (1973) 977107. 21. Mosher K. M., Young D. A. and Munck A.: Evidence for irreversible, actinomycin D-sensitive, and temperature-sensitive steps following binding of cortisol to glucocorticoid receptors and preceding effects on glucose metabolism in rat thymus cells. .f. biol. Chem. 246 (I 97 1) 654659. 22. Mueller R. E., Traish A. M. and Wotiz H. H.: Interaction of receptor-estrogen complex with uterine nuclei. J. biol. Chem. 252 (1977) 8206821 I. R. D.: Relationship of 23. Mulvihill E. R. and Palmiter nuclear estrogen receptor levels to induction of ovalbumin and conalbumin mRNA in chick oviduct. J. hiol. Chem. 252 (1977) 2060-2068.
than
complex. lation
one form
of “activated”
We are currently
of these
forms
trying
hormone to establish
to nuclear-bound
receptor the re-
complexes.
Ack,lowledgements-Supported by Research Grants AM03535, CA 17323, AM 20892 and Fellowship AM-05081, AM-20892
from the U.S. Public
Health
Service.
REFERENCES T.: Specific and nonI. Munck A. and Brinck-Johnsen specific physicochemical interactions of glucocorticoids and related steroids with rat thymus irl oirro. J. biol. Chum. 243 (1968) 55565565. 2. Wira C. R. and Munck A.: Glucocorticoid&eceptor complexes in rat thymus cells. Cytoplasmic-Nuclear Transformations. J. biol. Chem. 249 (1974) 5328-5336. receptors and 3. Munck A. and Leung K.: Glucocorticoid mechanisms of action. In Receptors and Mechanism of Action of Steroid Hormones (Edited by J. R. Pasqualini). Marcel Dekker, New York (1976) pp. 31 l-397, Part II. 4. Munck A. and Foley R.: Kinetics of glucocorticoidreceptor complexes in rat thymus cells. J. steroid Biothem. 7 (1976) II 17-l 122. K. R. and Alberts B. M.: Steroid receptors: 5. Yamamoto Elements for modulation of eukaryotic transcription. An. Rev. Biochem. 45 (1976) 721-744. 6. Spelsberg T. C., Pikler G. M. and Webster R. A.: Progesterone binding to hen oviduct genome: specific versus nonspecific binding. Science 194 (I 976) 197-l 98. 7. Spelsberg T. C., Steggles A. W. and O’Malley B. W.: Progesterone-binding components of chick oviduct III: “Chromatin Acceptor Sites”. J. hiol. Chern. 246 (1974) 41884197. of 8. Clark J. H. and Peck E. J. Jr: Nuclear retention receptor-oestrogen complex and nuclear acceptor sites. Nafure 260 (1976) 635-637. L., Chiu J, Tsai Y. and Hnilica 9. Klyzsehko-Stefanowicz L.: Acceptor proteins in rat androgenic tissue chromatin. Proc. narn. Acad. Sci.. U.S.A. 73 (1976) 19541958. IO. Simons S. S. Jr. Martinez H. M., Garcea R. L.. Baxter J. D. and Tomkins G. M.: Interactions of glucocorticoid receptor-steroid complexes with acceptor sites. J. hiol. Chem. 251 (1976) 334343.
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