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Non-stoichiometric nuclear-cytoplasmic redistribution of estrogen receptor in adult rat uterus, following estradiol injection
steroid Biochem.Vol. 24, No. 2, pp. 475479, Printed in Great Britain. All rights reserved
J.
1986 Copyright 0
0022-4731/86$3.00+ 0.00 1986 Pergamon Press Ltd
NON-STOICHIOMETRIC NUCLEAR-CYTOPLASMIC REDISTRIBUTION OF ESTROGEN RECEPTOR IN ADULT RAT UTERUS, FOLLOWING ESTRADIOL INJECTION E. EKKA, I. VANDERHEYDEN and R. DE HERTOGH* Physiology of Human Reproduction Research Unit, University of Louvain, UCL 5330. Avenue Em. Mounier, 53 B-1200 Brussels, Belgium (Received 13 June 1985) Summary-In immature and ovariectomized rats acutely injected with estradiol (&), accumulation of estradiol receptor complexes (E,R) from the uterine cytosol to the nucleus has been shown to be quantitative by numerous investigators. In the present study, translocation of E,R from the cytosol to the nuclear fraction in adult and ovariectomized estrogen prestimulated rats was analyzed. Twenty pg of E,, dissolved in saline containing 10% ethanol and 1 g% bovine serum albumin (B.S.A.) were injected intraperitoneally to the animals and 2 h later E,R in the cytosol and crude nuclear fractions were assayed by exchange techniques. Unlike a 91% recovery of the depleted cytosol QR in the nuclear fraction of ovariectomized rats, only 39.2 and 27.5% were recovered in the adult and ovariectomized estrogen prestimulated rat uterus respectively. Moreover, depending on the temperature and duration of nuclear suspension incubation, from 18 up’to 80% of the recovered nuclear&R were solubilized in the incubation medium and nuclear post-incubation washes and could be measured by hydroxylapatite treatment (HAP). Saturation assays showed a plateau from 12 nM E, ‘H onwards up to 80 nM. The Kd values computed for the receptors in the nucleus and HAP in all the three groups were of the order of 2 x 10e9M. In conclusion, after E, administration to adult or ovariectomized estrogen prestimulated rats, a stoichiometric recovery of the depleted cytosol E,R in the nuclear fraction was not observed, even when leakage of nuclear receptor into the medium in course of exchange was taken into account. Chronic estrogenization appeared to modify the dynamics of uterine receptor.
INTRODUCTION
Exchange assays are widely used for the measurement of cytosol [l] and nuclear [2] E,R in the rat uterus. Nuclear accumulation of depleted cytosol E,R observed after estradiol injection to immatue or ovariectomized animals was found quantitative by several authors[3-6]. However, in adult and in estrogen treated ovariectomized rats, the stoichiometric recovery was not observed [7 and present work]. Despite much speculation as regards the fate of the bound nuclear E,R, it still remains largely conjectural. Also, estrogen treatment may alter receptor dynamics [7, 81. Finally, a loss of receptor could arise from experimental procedures during the exchange assay [9, lo]. The aim of this study was to determine whether the discrepancy observed between cytosol and estrogen induced nuclear translocated E2R measurements resulted either from physiological decrease in receptor after estradiol administration to different animal models or was partially due to losses undergone during the exchange assay procedure. EXPERIMENTAL
Materials TWO to three month
breeding
center
old Wistar rats from the local
were
*To whom correspondence
bilaterally
ovariectomized
should be addressed.
and used 2-3 weeks later. Ovariectomized estrogen prestimulated rats received 5 pg E, subcutaneously in 0.25 ml saline containing 5% ethanol, for 2-4 consecutive days. Methods
Age matched adult (Ad), ovariectomized (Ox) and ovariectomized estrogen prestimulated (OxEP) rats were acutely injected intraperitoneally (i.p.) with 20 pg E, in 0.5 ml saline containing 10% ethanol and 1% BSA, 1 h before killing. Uteri were removed, cleaned of fat and mesentery and homogenized (1 uterus/ml) in ice-cold TEM buffer (10 mM Tris, I.5 mM EDTA and 14 mM mercaptoethanol, pH 7.4 at 20°C). Cytosol (lOS,OOOg, 30min) and crude nuclei @OOg, 3 washes) were prepared. Exchange assays were performed on cytosol [l] and nuclear pellet [2] as previously described [5]. To evaluate the losses of nuclear E,R during incubation and post-incubation nuclear washings, hydroxylapatite (HAP, Bio-gel H.T.P., Bio-Rad Lab., Calif., U.S.A.) assays [9, lo] were performed as follows: at the end of the nuclear pellet incubation, the medium was recovered, pooled with the three nuclear washes and added with 0.5 ml of HAP suspended in TEM (60% v/v). After vortexing and incubating at 0-4°C for 15 min, the HAP was recovered by centrifuging at 800 g for 10 mitt, and washed 475
E. EKKA et al.
476
thrice with 1 ml TEM at &4”C. Washed nuclear pellet and HAP were extracted with ethanol and counted. RESULTS
Table 1 shows the E,R distribution in cytosol, nuclear pellet and nuclear supernatant and washes (HAP) in the three groups of rats before and 1 h after i.p. injection of estradiol. The fraction of the cytosol E,R recovered in nuclei after injection (% recovered) is also indicated. A cumulated 91% of the depleted cytosol E,R was recovered in nuclei of Ox animals, whereas only 27.5 and 39.2% were recovered in OxEP and Ad respectively. From the total nuclear E2R, 18% were recovered from the incubation medium by HAP in Ox, 48% in OxEP and 33% in Ad animals. The importance of E,R leakage from the nuclear pellet to the medium led us to study more closely the effect of time and temperature of incubation on this potential loss. Table 2 shows a progressive increase of E,R leakage (HAP) with increasing incubation time, both at 37°C and at 20°C. For identical incubation times, a higher temperature induced increased leakage. Binding kinetics were analyzed on pellet associated sites as well as on incubation medium solubilized sites (HAP). Figures la, lb and lc show that saturation
obtained at a EZ3H concentration of 12nM in both fractions, in Ox (la), Ad (lb) and OxEP (Ic) animals. The K diss computed from the respective Scatchard plots showed similar values, in the range of 2nM (insets to Figs la, lb and Ic). Examination of two other tissue fractions (mitochondrial and microsomial fractions), discarded during subcellular fractionation, had no significant binding, since only negligable amount of radioactivity was recovered in these fractions after in gizmoinjection of Ez ‘H (personal observation). was
DISCUSSION
The present data indicate that after acute injection of E, to Ad and OxEP rats, the uterine E,R depleted from the cytosol was not recovered quantitatively in the nuclear fraction unlike what was observed in the Ox animals. Another characteristic of Ad and OxEP, as compared to Ox animals, was the more important leakage of nuclear E,R in the incubation medium in course of the exchange assay. These two observations together suggest biochemical differences in the nuclear-bound receptors of the different animal models, despite similar binding kinetics (Fig. la, lb, lc). The simi-
Table I. E,R distribution in ovariectomized (Ox), estrogen primed ovariectomized (OxEP) and adult non-ovariectomized (Ad) rat uteri, before (N. Inj.) and I b after i.p. injection (Inj.) of 2Opg E2 (pmol/ut. + SD) Nuclear fraction OX -N.
Inj.
-1j.
Cytosol
Pellet
HAP
% Recoveredt
I .20 + 0.57 (l2)* 0.13~0.11
0.10 f 0.05 (5) 0.90 * 0.30 (11)
0.03 * 0.01 (5) 0.2O~O.l2 (11)
91
(12) OxEP -N. Inj -1nj.
3.0(2) 0.31 (3)
0.16(2) 0.52 (2)
0.10(l) 0.48 (2)
2.05+ 0.72 (16) 0.29 rtO.12 (17)
0.27f 0.22 (6) 0.72 k 0.32 (18)
0.11 kO.09 (6) 0.35*0.12 (18)
27.5
Ad -N.
Inj
-1nj.
‘Number of experiments. tCalculated as follows: (Pellet + HAP) Ij. -(Pellet + HAP) N. Ij. Inj. - (Cytosol) Inj.
39.2
divided by (Cytosol) N.
Table 2. Effect of time and temperature of incubation on nuclear distribution of E,R between pellet and HAP Temperature (“C) Time (h) OX -Total (pmollut) -Pellet (%) -HAP (%)
37 l/4 0.89 82 18
37 l/2 0.99 76 24
37
31
20
20
0
I
2
I
24
24
1.10
1.01
64 36
57 43
0.44 74 26
1.14 34 66
Ad
-Total (pmoliut) -Pellet (%) -HAP (%)
1.23 89 II
1.14 83 I7
I .05 75 25
I .42 74 26
0.34 86 14
1.11 23 77
0.80 71 29
E,R transiocation in rat uterine nuclei
ox. A
C
l3
2.1 2.2
19 nM
50 nM
Ad.
pmolfut.
A i.2.09
0
C
1.76
207nM
B/F
L
0
10
20
30
CO
,
,
50
60
?OnM
ox.EP
10
20
A
50
60
B
C
70nM
Cc) Fig. la, lb, le. Saturation curves of specific binding and Scatchard plot analysis performed on crude nuclear suspensions of (la) Ox, (lb) Ad and (1~) OxEP rat uteri. Nuclear suspensions were prepared as described in Experimental. 200 pl of the nuclear suspension containing 0.75 ug/ml in TEM buffer were incubated for 1 h at 0-4°C followed by 1h incubation at 37°C in the presence of varying concentrations ranging from 0.5 to 40 nM of Ez 3H alone or in the presence of l~foid excess of DES. Specific binding was calculated by subtracting the non-specific binding from the total binding. Bach point is the mean of triplicates: A. nuclear pellets. B. HAP recuperated E2R from pooled post-incubation medium and three post-incubation nuclear washes. C. cumulated (pellet + HAP) curve. Insets: Scatchard plot analysis of the three saturation curves for each group.
478
E. EKKA et al.
larity between Ad and OxEP in opposition to Ox animals suggests also a role of estrogen priming in these observations. A similar discrepancy in the nuclear receptor recovery of the depleted cytosol receptors has been reported by Walters et al.[l 1] for progesterone in OxEP animals, and attributed to solubilization of progesterone receptor complex into the supernatant and to ligand-receptor dissociation. These authors however observed insignificant leakage of E2R from nuclei of immature rats [9]. Several hypotheses have been advanced to explain nuclear E,R losses in different experimental models. Horwitz et a1.[12, 131 and Strobl et a1.[14] reported a decline in nuclear receptor levels, termed “processing” after incubation of human breast cancer MCF-7 cells with Ez in vitro. The mechanism involved appeared complex, including both kinetic changes in receptor properties and loss of binding capacity[14]. Kassis et a/.[151 and Pavlik et a/.[161 also refer to the “processing” mechanism to account for E,R losses in the rats receiving an injection of a long-acting estrogen like EZand also in rat uterine cell suspension. The “processing” was attributed to reversible inactivation of the binding capacity of the receptor [ 151. No processing, but time-dependent changes in receptor physico-chemical properties was described by Jackesz et a/.[171 in rat uterus subjected to continuous E, exposure. In in vitro observations, Auricchio et al.[l8] attributed the loss in nuclear E,R
to an inactivation process by dephosphorylation. Continuous exposure of uteri to estrogen has been shown by Sato et a[.[71 to alter the regulation of estrogen receptor dynamics, very much like the observation reported in the present study. The mechanism by which E,R dynamics are modified by estrogenization is presently not known and probably complex [7,8]. The nature of “processing” remains also unclear. It may be an active state in which a new equilibrium between degradation and synthesis is achieved [14, 19,201 or a redistribution of receptor within nuclear binding sites of different affinities or specificities, or sequestration of receptor to sites inaccessible to exchange [21-231. E,R attachment to the nuclear matrix has recently been shown by Barrack et a1.[24]. In this eventuality, the accessibility and differential retention of nuclear E,R, so also the exchangeability could be profoundly modified under the effect of E, and could be reflected as a discrepancy in the measured nuclear E2R with conventional exchange assays. In conclusion, stoichiometric recovery in the
nuclear fraction of cytosol E,R translocated under the influence of acute Ez injection is not a genera1 event and is linked to the state of estrogenization of the rat. Chronic estrogen administration appear to reduce nuclear recovery, without any change in binding kinetics of the recovered sites. A significant proportion of these sites appears to leak from the nuclei into the medium in course of the incubation
procedure, depending on time and temperature of incubation and on estrogen priming of the animal. Exchange methods should take this leakage into consideration to avoid supplementary loss of binding sites. Acknou~ledgements-This
work was supported by a grant of the Fonds de la Recherche Scientifique Mddicale of Belgium. The skilfull technical assistance of B. Glorieux was much appreciated. REFERENCES I.
Katzenellenbogen J. A., Johnson H. J. Jr and Carlson K. E.: Studies on the uterine, cytoplasmic estrogen binding protein. Thermal stability and ligand dissociation rate. An assay of empty and filled sites by exchange. Biochemistry 12 (1973) 40924099. 2. Anderson J., Clark J. H. and Peck E. J. Jr: Oestrogen and nuclear binding sites. Determination of soecific sites by [3H] oestradiol-exchange. Biochem. J. i26 (1972) 561-567.
3 Shyamala G. and Gorski J.: Estrogen receptors in the rat uterus. Studies on the interaction of cytosol and nuclear binding sites. J. biol. Chem. 244 (1969) 1097-I 103.
4 Giannopoulos G. and Gorski J.: Estrogen receptors. Quantitative studies on transfer of estradiol from cytoplasmic to nuclear binding sites. J. biol. Chem. 246 (1971) 25242529. 5. De Hertogh R., Ekka E. and Vanderheyden I.: Estrogen receptor and stimulation of uterine protein synthesis in ovariectomized diabetic rats infused intravenously with 17/3-estradiol. Endocrinology 110 (1982) 741-748. 6. Ekka E., Vanderheyden I. and De Hertogh R.: Short term nuclear retention and early cytosol replenishment of estradiol receptors in uteri of ovariectomized diabetic rats after intraperitoneal injection of l7b-estradiol: evidence for decreased hormonal activity on protein synthesis. Endocrinology 111 (I 982) 45646 I. 7. Sato B., Nishizawa Y., Noma K., Kishimoto S. and Matsumoto K.: Chronic estrogen treatment causes an alteration in uterine estrogen receptor dynamics of rats. Biochim. biophys. Acta 755 (1983) 412-419.
8. Cidlowski J. A. and Multoon T. G.: The dynamics of intracellular estrogen receptor regulation as influenced by l7/?-estradiol. Biol. Reprod. 18 (1978) 234-246. 9. Walters M. R., Hunziker W. and Clark J. H.: Hydroxylapatite prevents nuclear receptor loss during the exchange assay of progesterone receptors. J. steroid Biochem. 13 (1980) 1129-l 132.
10. Pavlik E. J. and Coulson P. B.: Hydroxylapatite “batch” assay for estrogen receptor: increased sensitivity over present receptor assay. J. steroid Biochem. 7 (1976) 357-368.
I1 Walters M. R. and Clark J. H.: Stoichiometric translocation of the rat uterine progesterone receptor. Endocrinolopv 103 (1978) 1952-1955.
12 Horwitz K.-B. and’ Mcduire W. L.: Estrogen control of progesterone receptor in human breast cancer. Correlation with nuclear processing of estrogen receptor. J. biol. Chem. 253 (1978) 2223-2228. 13. Horwitz K. B. and McGuire W. L.: Nuclear estrogen receptors. Effect of inhibitors on processing and steady state levels. J. biol. Chem. 255 (1980) 9699-9705. 14. Strobl J. S., Kasid A., Huff K. K. and Lippman M. E.: Kinetic alterations in estrogen receptors associated with estrogen receptor processing in human breast cancer cells. Endocrinology 115 (1984) 11161124. I5 Kassis J. A. and Gorski J.:’ On the mechanism of estrogen receptor replenishment, recycling, resynthesis and/or processing. Molec. cell Eiochem. 52 (1983) 27-36.
E,R translocation 16. Pavlik E. J., Rutledge G., Eckert R. L. and Katzenellenbogen B. S.: Localization of estrogen receptors in uterine cells. Expl. ceil. Res. 123 (1979) 177-189. 17. Jackesz R., Kasid A., Greene G. and Lippman M. E.: Characteristics of different cytoplasmic and nuclear receptors appearing with continuous hormonal exposure. J. biol. Chem. 258 (1983) 11807-11813. 18. Auricchio F.. Migliaccio A. and Rotondi A.: Inactivation of estrogen receptor in vitro by nuclear dephosphorylation. Biochem. J. 194 (1981) 569-574. 19. Sarff M. and Gorski J.: Control of estrogen binding protein concentration under basal conditions and after estrogen administration. Biochemistry 10 (1971) 255172563. 20. Williams D. and Gorski J.: Kinetics and equilibrium
in rat uterine nuclei
419
analysis of estradiol in uterus. A model of binding site distribution in uterine cells. Proc. natn. Acad. Sci. U.S.A. 69 (1972) 3464-3468. 21. De Hertogh R., Ekka E., Vanderheyden
J. J.: Slowly exchangeable uterus. J. steroid Biochem. 22. Clark J. H. and Peck E. receptor estrogen complex
I. and Hoet pool of estradiol in the rat 4 (1973) 3 13-320. J. Jr: Nuclear retention of and nuclear receptor sites.
Nature 260 (1976) 635-637. 23. Ruh T. S. and Baudendistel
L. J.: Different nuclear binding sites for antiestrogen and estrogen receptor complexes. Endocrinology 100 (1977) 42W-426. 24. Barrack E. R. and Coffey D. S.: Biological properties of the nuclear matrix: steroid hormone binding. Recent Prog. Harm. Res. 38 (1982) 133-195.
J.
1986 Copyright 0
0022-4731/86$3.00+ 0.00 1986 Pergamon Press Ltd
NON-STOICHIOMETRIC NUCLEAR-CYTOPLASMIC REDISTRIBUTION OF ESTROGEN RECEPTOR IN ADULT RAT UTERUS, FOLLOWING ESTRADIOL INJECTION E. EKKA, I. VANDERHEYDEN and R. DE HERTOGH* Physiology of Human Reproduction Research Unit, University of Louvain, UCL 5330. Avenue Em. Mounier, 53 B-1200 Brussels, Belgium (Received 13 June 1985) Summary-In immature and ovariectomized rats acutely injected with estradiol (&), accumulation of estradiol receptor complexes (E,R) from the uterine cytosol to the nucleus has been shown to be quantitative by numerous investigators. In the present study, translocation of E,R from the cytosol to the nuclear fraction in adult and ovariectomized estrogen prestimulated rats was analyzed. Twenty pg of E,, dissolved in saline containing 10% ethanol and 1 g% bovine serum albumin (B.S.A.) were injected intraperitoneally to the animals and 2 h later E,R in the cytosol and crude nuclear fractions were assayed by exchange techniques. Unlike a 91% recovery of the depleted cytosol QR in the nuclear fraction of ovariectomized rats, only 39.2 and 27.5% were recovered in the adult and ovariectomized estrogen prestimulated rat uterus respectively. Moreover, depending on the temperature and duration of nuclear suspension incubation, from 18 up’to 80% of the recovered nuclear&R were solubilized in the incubation medium and nuclear post-incubation washes and could be measured by hydroxylapatite treatment (HAP). Saturation assays showed a plateau from 12 nM E, ‘H onwards up to 80 nM. The Kd values computed for the receptors in the nucleus and HAP in all the three groups were of the order of 2 x 10e9M. In conclusion, after E, administration to adult or ovariectomized estrogen prestimulated rats, a stoichiometric recovery of the depleted cytosol E,R in the nuclear fraction was not observed, even when leakage of nuclear receptor into the medium in course of exchange was taken into account. Chronic estrogenization appeared to modify the dynamics of uterine receptor.
INTRODUCTION
Exchange assays are widely used for the measurement of cytosol [l] and nuclear [2] E,R in the rat uterus. Nuclear accumulation of depleted cytosol E,R observed after estradiol injection to immatue or ovariectomized animals was found quantitative by several authors[3-6]. However, in adult and in estrogen treated ovariectomized rats, the stoichiometric recovery was not observed [7 and present work]. Despite much speculation as regards the fate of the bound nuclear E,R, it still remains largely conjectural. Also, estrogen treatment may alter receptor dynamics [7, 81. Finally, a loss of receptor could arise from experimental procedures during the exchange assay [9, lo]. The aim of this study was to determine whether the discrepancy observed between cytosol and estrogen induced nuclear translocated E2R measurements resulted either from physiological decrease in receptor after estradiol administration to different animal models or was partially due to losses undergone during the exchange assay procedure. EXPERIMENTAL
Materials TWO to three month
breeding
center
old Wistar rats from the local
were
*To whom correspondence
bilaterally
ovariectomized
should be addressed.
and used 2-3 weeks later. Ovariectomized estrogen prestimulated rats received 5 pg E, subcutaneously in 0.25 ml saline containing 5% ethanol, for 2-4 consecutive days. Methods
Age matched adult (Ad), ovariectomized (Ox) and ovariectomized estrogen prestimulated (OxEP) rats were acutely injected intraperitoneally (i.p.) with 20 pg E, in 0.5 ml saline containing 10% ethanol and 1% BSA, 1 h before killing. Uteri were removed, cleaned of fat and mesentery and homogenized (1 uterus/ml) in ice-cold TEM buffer (10 mM Tris, I.5 mM EDTA and 14 mM mercaptoethanol, pH 7.4 at 20°C). Cytosol (lOS,OOOg, 30min) and crude nuclei @OOg, 3 washes) were prepared. Exchange assays were performed on cytosol [l] and nuclear pellet [2] as previously described [5]. To evaluate the losses of nuclear E,R during incubation and post-incubation nuclear washings, hydroxylapatite (HAP, Bio-gel H.T.P., Bio-Rad Lab., Calif., U.S.A.) assays [9, lo] were performed as follows: at the end of the nuclear pellet incubation, the medium was recovered, pooled with the three nuclear washes and added with 0.5 ml of HAP suspended in TEM (60% v/v). After vortexing and incubating at 0-4°C for 15 min, the HAP was recovered by centrifuging at 800 g for 10 mitt, and washed 475
E. EKKA et al.
476
thrice with 1 ml TEM at &4”C. Washed nuclear pellet and HAP were extracted with ethanol and counted. RESULTS
Table 1 shows the E,R distribution in cytosol, nuclear pellet and nuclear supernatant and washes (HAP) in the three groups of rats before and 1 h after i.p. injection of estradiol. The fraction of the cytosol E,R recovered in nuclei after injection (% recovered) is also indicated. A cumulated 91% of the depleted cytosol E,R was recovered in nuclei of Ox animals, whereas only 27.5 and 39.2% were recovered in OxEP and Ad respectively. From the total nuclear E2R, 18% were recovered from the incubation medium by HAP in Ox, 48% in OxEP and 33% in Ad animals. The importance of E,R leakage from the nuclear pellet to the medium led us to study more closely the effect of time and temperature of incubation on this potential loss. Table 2 shows a progressive increase of E,R leakage (HAP) with increasing incubation time, both at 37°C and at 20°C. For identical incubation times, a higher temperature induced increased leakage. Binding kinetics were analyzed on pellet associated sites as well as on incubation medium solubilized sites (HAP). Figures la, lb and lc show that saturation
obtained at a EZ3H concentration of 12nM in both fractions, in Ox (la), Ad (lb) and OxEP (Ic) animals. The K diss computed from the respective Scatchard plots showed similar values, in the range of 2nM (insets to Figs la, lb and Ic). Examination of two other tissue fractions (mitochondrial and microsomial fractions), discarded during subcellular fractionation, had no significant binding, since only negligable amount of radioactivity was recovered in these fractions after in gizmoinjection of Ez ‘H (personal observation). was
DISCUSSION
The present data indicate that after acute injection of E, to Ad and OxEP rats, the uterine E,R depleted from the cytosol was not recovered quantitatively in the nuclear fraction unlike what was observed in the Ox animals. Another characteristic of Ad and OxEP, as compared to Ox animals, was the more important leakage of nuclear E,R in the incubation medium in course of the exchange assay. These two observations together suggest biochemical differences in the nuclear-bound receptors of the different animal models, despite similar binding kinetics (Fig. la, lb, lc). The simi-
Table I. E,R distribution in ovariectomized (Ox), estrogen primed ovariectomized (OxEP) and adult non-ovariectomized (Ad) rat uteri, before (N. Inj.) and I b after i.p. injection (Inj.) of 2Opg E2 (pmol/ut. + SD) Nuclear fraction OX -N.
Inj.
-1j.
Cytosol
Pellet
HAP
% Recoveredt
I .20 + 0.57 (l2)* 0.13~0.11
0.10 f 0.05 (5) 0.90 * 0.30 (11)
0.03 * 0.01 (5) 0.2O~O.l2 (11)
91
(12) OxEP -N. Inj -1nj.
3.0(2) 0.31 (3)
0.16(2) 0.52 (2)
0.10(l) 0.48 (2)
2.05+ 0.72 (16) 0.29 rtO.12 (17)
0.27f 0.22 (6) 0.72 k 0.32 (18)
0.11 kO.09 (6) 0.35*0.12 (18)
27.5
Ad -N.
Inj
-1nj.
‘Number of experiments. tCalculated as follows: (Pellet + HAP) Ij. -(Pellet + HAP) N. Ij. Inj. - (Cytosol) Inj.
39.2
divided by (Cytosol) N.
Table 2. Effect of time and temperature of incubation on nuclear distribution of E,R between pellet and HAP Temperature (“C) Time (h) OX -Total (pmollut) -Pellet (%) -HAP (%)
37 l/4 0.89 82 18
37 l/2 0.99 76 24
37
31
20
20
0
I
2
I
24
24
1.10
1.01
64 36
57 43
0.44 74 26
1.14 34 66
Ad
-Total (pmoliut) -Pellet (%) -HAP (%)
1.23 89 II
1.14 83 I7
I .05 75 25
I .42 74 26
0.34 86 14
1.11 23 77
0.80 71 29
E,R transiocation in rat uterine nuclei
ox. A
C
l3
2.1 2.2
19 nM
50 nM
Ad.
pmolfut.
A i.2.09
0
C
1.76
207nM
B/F
L
0
10
20
30
CO
,
,
50
60
?OnM
ox.EP
10
20
A
50
60
B
C
70nM
Cc) Fig. la, lb, le. Saturation curves of specific binding and Scatchard plot analysis performed on crude nuclear suspensions of (la) Ox, (lb) Ad and (1~) OxEP rat uteri. Nuclear suspensions were prepared as described in Experimental. 200 pl of the nuclear suspension containing 0.75 ug/ml in TEM buffer were incubated for 1 h at 0-4°C followed by 1h incubation at 37°C in the presence of varying concentrations ranging from 0.5 to 40 nM of Ez 3H alone or in the presence of l~foid excess of DES. Specific binding was calculated by subtracting the non-specific binding from the total binding. Bach point is the mean of triplicates: A. nuclear pellets. B. HAP recuperated E2R from pooled post-incubation medium and three post-incubation nuclear washes. C. cumulated (pellet + HAP) curve. Insets: Scatchard plot analysis of the three saturation curves for each group.
478
E. EKKA et al.
larity between Ad and OxEP in opposition to Ox animals suggests also a role of estrogen priming in these observations. A similar discrepancy in the nuclear receptor recovery of the depleted cytosol receptors has been reported by Walters et al.[l 1] for progesterone in OxEP animals, and attributed to solubilization of progesterone receptor complex into the supernatant and to ligand-receptor dissociation. These authors however observed insignificant leakage of E2R from nuclei of immature rats [9]. Several hypotheses have been advanced to explain nuclear E,R losses in different experimental models. Horwitz et a1.[12, 131 and Strobl et a1.[14] reported a decline in nuclear receptor levels, termed “processing” after incubation of human breast cancer MCF-7 cells with Ez in vitro. The mechanism involved appeared complex, including both kinetic changes in receptor properties and loss of binding capacity[14]. Kassis et a/.[151 and Pavlik et a/.[161 also refer to the “processing” mechanism to account for E,R losses in the rats receiving an injection of a long-acting estrogen like EZand also in rat uterine cell suspension. The “processing” was attributed to reversible inactivation of the binding capacity of the receptor [ 151. No processing, but time-dependent changes in receptor physico-chemical properties was described by Jackesz et a/.[171 in rat uterus subjected to continuous E, exposure. In in vitro observations, Auricchio et al.[l8] attributed the loss in nuclear E,R
to an inactivation process by dephosphorylation. Continuous exposure of uteri to estrogen has been shown by Sato et a[.[71 to alter the regulation of estrogen receptor dynamics, very much like the observation reported in the present study. The mechanism by which E,R dynamics are modified by estrogenization is presently not known and probably complex [7,8]. The nature of “processing” remains also unclear. It may be an active state in which a new equilibrium between degradation and synthesis is achieved [14, 19,201 or a redistribution of receptor within nuclear binding sites of different affinities or specificities, or sequestration of receptor to sites inaccessible to exchange [21-231. E,R attachment to the nuclear matrix has recently been shown by Barrack et a1.[24]. In this eventuality, the accessibility and differential retention of nuclear E,R, so also the exchangeability could be profoundly modified under the effect of E, and could be reflected as a discrepancy in the measured nuclear E2R with conventional exchange assays. In conclusion, stoichiometric recovery in the
nuclear fraction of cytosol E,R translocated under the influence of acute Ez injection is not a genera1 event and is linked to the state of estrogenization of the rat. Chronic estrogen administration appear to reduce nuclear recovery, without any change in binding kinetics of the recovered sites. A significant proportion of these sites appears to leak from the nuclei into the medium in course of the incubation
procedure, depending on time and temperature of incubation and on estrogen priming of the animal. Exchange methods should take this leakage into consideration to avoid supplementary loss of binding sites. Acknou~ledgements-This
work was supported by a grant of the Fonds de la Recherche Scientifique Mddicale of Belgium. The skilfull technical assistance of B. Glorieux was much appreciated. REFERENCES I.
Katzenellenbogen J. A., Johnson H. J. Jr and Carlson K. E.: Studies on the uterine, cytoplasmic estrogen binding protein. Thermal stability and ligand dissociation rate. An assay of empty and filled sites by exchange. Biochemistry 12 (1973) 40924099. 2. Anderson J., Clark J. H. and Peck E. J. Jr: Oestrogen and nuclear binding sites. Determination of soecific sites by [3H] oestradiol-exchange. Biochem. J. i26 (1972) 561-567.
3 Shyamala G. and Gorski J.: Estrogen receptors in the rat uterus. Studies on the interaction of cytosol and nuclear binding sites. J. biol. Chem. 244 (1969) 1097-I 103.
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