Effect of age on physico-chemical properties of the uterine nuclear estrogen receptors of albino rats

Mechanisms of Ageing and Development, 57 (1991) 111-- 123

111

ElsevierScientificPublishersIrelandLtd.

EFFECT OF AGE ON PHYSICO-CHEMICAL PROPERTIES OF T H E UTERINE NUCLEAR ESTROGEN RECEPTORS OF ALBINO RATS

J. KAUR and M.K. THAKUR Biochemistry and Molecular Biology Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005 (India)

(ReceivedMay5th, 1990)

SUMMARY Estrogen receptors (ER) were extracted with high ionic strength buffer from the nuclei of uteri of young (21 weeks) and old (89 weeks) rats. Following the analysis of these receptors on a sucrose gradient and a Sephadex column, two peaks representing the two forms of receptors were obtained. The minor peak sedimented at 6.8 S with Stokes radius (Rs) = 7.2 nm, molecular weight (Mr) = 204 K and frictional ratio (f/fo) = 1.71. On the other hand, the major peak sedimented at 4.1 S with R s = 3.3 nm, Mr = 57 K a n d f / f 0 = 1.18. These properties of nuclear ER were similar in both ages. Also, the half life of ER complexes from both ages at 25 °C and 37 °C were 135 rain and 30 min, respectively. However, these complexes were retained for longer periods in the nuclei of young than old rats. Furthermore, the dissociation constant of the binding of nuclear receptors to estrogen remained constant, but the number of binding sites decreased from 1.56 in young to 1.05 pmol/mg DNA in the old. In young rats, about 61% of nuclear receptors bound to DNA-cellulose. Out of this 2/5 was eluted with 0.15 M and the remaining 3/5 with 0.5 M KCI. On contrary, only 37eTeof total receptors bound to DNA-cellulose in the old. Out of this 3/5 was eluted with 0.15 M and the remaining 2/5 with 0.5 M KCI. These data suggested that despite the similarity in different physicochemical properties, the number of estrogen binding sites and the retention time of ER complexes in nuclei and the ability of these complexes to bind to DNA decrease in the uterus of old age.

K e y words: Physico-chemical properties; Nuclear estrogen receptor; Aging (rat

uterus)

Correspondence to: M.K. Thakur, Biochemistryand Molecular BiologyLaboratory, Department of Zoology,BanarasHinduUniversity,Varanasi221 005, India.

0047-6374/91/$03.50 Printedand Publishedin Ireland

© 1991ElsevierScientificPublishersIrelandLtd.

112 INTRODUCTION

The concept of an initial interaction between estrogen and its cognate receptor in the cytoplasm followed by translocation of the resultant complex to the nuclei has been questioned during the past few years. Now substantial evidences have accumulated indicating that ER reside inside the nuclei of estrogen responsive cells [1--3]. Some of these receptors are loosely bound to chromatin. They get released into cytosol during tissue disruption and are designated as cytoplasmic receptors. In contrast, some receptors are tightly associated with chromatin. They are present in partially purified nuclear pellet and are termed nuclear receptors. Several studies have been reported on age-dependent decline in the level of both cytoplasmic and nuclear ER of the rat uterus [4,5]. Also the rate of transformation of these receptors as well as their binding to nuclei decrease in old age [6,7]. A variety of biochemical approaches have been used to determine the molecular properties of ER [8]. However, it is not clearly understood whether the physico-chemical characteristics of these receptors change with age. Therefore, it is reasonable to compare physico-chemical properties of nuclear ER between the uterus of young and old rats. In the present investigation, sedimentation coefficient (S) was determined by sucrose gradient centrifugation, and Stokes radius (Rs) by gel filtration. Molecular weight (Mr) and frictional ratio (fifo) were calculated from S and R s values. The half life (tl/2) of ER complexes and their retention time were determined by exchange assay. Furthermore, an attempt was made to examine whether the binding of nuclear receptors to 17/J-estradiol and the binding of ER complexes to DNA alter during aging. MATERIALS AND METHODS

,4 nimals and chemicals Young (21 weeks) and old (89 weeks) female albino rats of Wistar strain (Rattus norvegicus) maintained under standard colony conditions were used. They were ovariectomized under light ether anesthesia. After 3 weeks of the operation, they were injected with 5/ag 17/J-estradiol/100 g body wt. The rats were killed after 1 h and the uteri were removed and stripped of fat and connective tissues. [3HlEstradiol (spec. act. 88 Ci/mmol) was purchased from Amersham, U.K. Celo lexo410 was obtained from Bio-Rad Laboratories, U.S.A. All other chemicals were bought mainly from Sigma Chemical Co., St. Louis, U.S.A.

Preparation o f nuclear ER ER were prepared from the uterine nuclei of rats treated previously with unlabeled estradiol. All steps were carried out at 0--4° C unless otherwise specified. The uteri were homogenized using a VirTis 45 homogenizer in 4 vols. of TEDG (20 mM Tris--I-IC1/1.5 mM EDTA/1 mM DTT/10°70 glycerol) buffer (pH 7.4), containing 1

113 mM PMSF. The homogenate was centrifuged at 800 g for 15 min. The resulting crude nuclear pellet was washed twice with T E D G buffer. To extract nuclear receptors, the pellet was suspended in 4 vols. of T E D G buffer (pH 8.0), containing 0.5 M KCI (TEDGK). The nuclear suspension was left on ice for 1 h with intermittent vortexing at 15-min intervals. It was centrifuged at 100 000 g for 30 min and the supernatant was saved as nuclear receptors. The receptors were incubated with 20 nM [3H]estradiol in the presence or absence o f 4/~M unlabeled estradiol at 30°C for 1 h. Then it was transferred onto ice. The extent of specifically bound estradiol was calculated as the difference between total and non-specific binding (measured in the absence or presence of unlabeled estradiol, respectively).

Sucrose gradient centrifugation Nuclear ER (0.4 ml) from both ages were adjusted to approximately equal radioactivity and layered on a 4.8 ml linear (5--20070) sucrose gradient prepared in T E D G K buffer. Horse Radish Peroxidase (3.6 S) and glucose oxidase (7.9 S) were included in the sample as internal standards. The tubes were centrifuged in a Beckman VTi 80 rotor at 50 000 rev./min for 2 h. Fractions (150/~l) were collected from the bottom of the tubes and the radioactivity was counted. Each fraction was also assayed for enzyme activity.

Gelfiltration analysis Preswollen Sephadex G-200 was packed into a 1 x 37 cm column and equilibrated at 4°C with TEDGK buffer containing 0.1°70 BSA. A 1.5 ml aliquot of the nuclear extract was loaded on the column and eluted with TEDGK buffer at a flow rate of 3 ml/h. Fractions of 1 ml were collected and the radioactivity was counted. The protein standards used to calibrate the column were: ferritin, BSA, ovalbumin and cytochrome c. The distribution coefficient (Ko) was calculated according to the equation K D = (V - Vo/(Vt - V0) where V0, the void volume was determined with blue Dextran 2000; Vt, the total volume of the gel bed was measured with potassium dichromate; and V is the elution volume of the receptor or the standard proteins.

Molecular weight, frictional ratio and half life determination M and f/fo of the nuclear ER were calculated with the help of S and R s values according to Siegel and Monty [9]. In order to find out t~/2, the labeled ER was first treated with dextran-coated charcoal (DCC) and then incubated in the presence or absence o f 4/~M unlabeled estradiol at 25 °C and 37 °C. At different time intervals (15 min - - 3 h), the aliquots were removed, treated with DCC and counted for radioactivity.

Kinetics of estradiol binding The level o f nuclear ER was determined according to the exchange method of

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Anderson et al. [10]. The 800 g nuclear pellet was suspended in TED buffer (pH 7.5). Aliquots of this suspension (125/~g DNA) were dispensed in tubes containing 1--20 nM [3H]estradiol with or without 100-fold unlabeled estradiol in 0.4 ml. Following incubation at 37 °C for 30 min, the tubes were centrifuged and the pellet was washed thrice with cold TED buffer. It was extracted with 1 ml absolute ethanol and counted for radioactivity with 33°70 efficiency. The specific binding was calculated by subtracting non-specific binding from the total binding. Dissociation constant and the number of binding sites were calculated by Scatchard plot [ 11].

Retention time o f ER complexes Ovariectomized young and old rats were injected with 17/3-estradiol and killed after different time periods 05 min--6 h). Uterine nuclei were isolated and assayed by exchange method of Anderson et al. [10]. The number of specifically bound ER complexes were calculated. The [3H]estradiol labeled nuclei were further extracted with buffer containing high salt (TEDGK). The salt-resistant fractions were analysed for determining the number of tightly bound nuclear ER complexes. DNA-cellulose chromatography DNA-cellulose was prepared using calf thymus DNA according to Alberts and Herrick [12]. [3H]Estradiol-labeled nuclear extract was dialysed against TEDG buffer and mixed with DNA-cellulose (0.9 mg DNA/g cellulose). The mixture was incubated for 90 rain at 4°C with intermittent mixing and packed into a 1.0 x 3.2 cm column. The column was washed with TEDG buffer until the radioactivity in the washing reached a low constant background and then eluted stepwise first with buffer containing 0.15 M KCI and later with buffer containing 0.5 M KCI. Fractions of 0.5 ml wre collected and the radioactiity was counted. RESULTS

Sedimentation behaviour Figure 1 shows the sedimentation profile of the ER extracted with 0.5 M KC1 from the uterine nuclei of young and old rats. Two peaks could be seen following sedimentation of this fraction on a sucrose gradient. The minor peak sedimented at 6.8 S and the major peak representing slowly sedimenting form at 4.1 S. This is similar in both ages. However, the incorporation of radioactivity differs in young rats as compared to the old. In the minor peak, there is 3-fold higher incorporation in old age. On the other hand, young rats show nearly double incorporation in the major peak. Gel chromatography analysis The elution pattern of receptors and the position of protein standards are shown in Fig. 2 (top) and the column calibration line in Fig. 2 (bottom). The Stokes radius

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25

~

--

20

x

z o I-- 1 - 5

u .<

II. Q

5

0

I

I

10

20

30

Bottom

Top FRACTION

No.

Fig. 1. Sedimentation profile of pH]estradiol-labeled nuclear ER o f the uteri of young (O O) and old ( x × ) rats on 5--20°70 sucrose gradient. Arrows indicate the positions of horseradish peroxidase (3.6 S) and glucose oxidase (7.9 S) used as internal markers.

of the receptor was estimated by comparing its elution pattern on the column with that of other standard proteins whose Stokes radii are known. The receptor got eluted from the column as two peaks - - one minor peak close behind ferritin with an estimated Stokes radius of 7.2 nm and another major peak between BSA and ovalbumin with an estimated Stokes radius of 3.3 nm. The values of R s remain similar in both ages.

Molecular weight, frictional ratio and half life determination Utilizing the Stokes radii estimated by Sephadex G-200 chromatography and the sedimentation coefficients determined from sucrose gradient analysis, the molecular weights and frictional ratios were calculated by the equation of Siegel and Monty [9], and are summarized in Table Ia. The minor peak had a calculated M of about 204 K and a f/fo of 1.72 whereas the major peak had a M of 57 K and a f / f o of 1.19. The M andf/fo calculated for the receptor solubilized by 0.5 M KCI were identical in both ages. The half life of nulcear ER complexes from both ages determined at 25°C and 37°C were 135 min and 30 min, respectively (Fig. 3).

116

2o!

Vo F 12

BSA OV 34

,

z O

CC V e 56

9

15

k-

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I 16

0

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FRACTION

32

No.

1'2

0'8 v VO. 4

'

!

2

i

I

,

4

l

6

,

I

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8

R$ ( r i m )

Fig. 2. Estimation of Stokes radii of nuclear ER by Sephadex G-200 column chromatography. Nuclear ER were prepared as described under Experimental and eluted through a Sephadex column in T E D G K buffer. Ferritin (F, R s = 7.0 rim), BSA (R s = 3.55 nm), Ovalbumin (Ov, R s = 2.9 nm) and Cytochrome c (CC, R s = 1.64 nm) were chromatographed as standards. Top, the elution profile of nuclear ER from young (O O) and old ( x × ) rats. Bottom, a calibration line showing the relationship between Stokes radii (Rs) and distribution coefficients (Ko) for the protein standards and the receptors (marked with arrows).

Kinetics of estrogen binding The binding o f 17/3-estradiol to ER was relatively higher in young rats as compared to the old (Fig. 4a). The dissociation constant was almost similar about 1.20 nm in young and 1.34 nm in old (Fig. 4b). However, the number of binding sites

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TABLE I (A) P H Y S I C O - C H E M I C A L P R O P E R T I E S OF FAST (I) A N D S L O W L Y (II) S E D I M E N T I N G FORMS O F N U C L E A R ER O F Y O U N G A N D O L D RATS

Parameters

Young

Sedimentation coefficients (S) Stokes radius (nm) Frictionalratio Molecular weight

Old

I

H

I

H

6.80 ± 0.05

4.10 ± 0.15

6.70 ± 0.09

3.90 ± 0.11

7.20 ± 0.12

3.30 ± 0.02

7.30 ± 0.07

3.50 ± 0.14

1.71 ± 0.02 204 ± 3.47

1.18 ± 0.01 57 ± 0.85

1.73 ± 0.04 206 ± 3.30

1.20 ± 0.06 57 ± 1.44

(I<) (B) KINETIC ANALYSIS OF T H E BINDING OF [3H]ESTRADIOL TO N U C L E A R ER OF Y O U N G A N D O L D RATS

Parameters

Young

Old

Dissociation constant (nM) Number of binding sites ( p m o l / m g DNA)

1.20 ± 0.02

1.34 ± 0.12

1.56 ± 0.16

1.05 ± 0.19"

The data were collected from three independent sets o f experiments. Values are means ± S.D. Significance o f differences between young and old group is indicated by *P< 0.05.

.Z

100

CJ

,'7

50

.,< 0

,

I

1

I

i

1

2

3

4

TIME

( h )

Fig. 3. Determination o f half life of nuclear ER from the uteri o f y o u n g (O O) and old × ) rats. The release o f radioactivity from pH]estradiol-labeled ER was measured at 25 °C and (x 37°C.

118

® o~1.0 E

o YOUNG

o

E

= OLD

uJ 0.5 -r o z o ii1 I

I

Io 20 [3HI Est CONC. {nM) 0.4

0.3

0.2

0.1 Q

0,5

1.0

1"5

BOUND [3HI Est (P mole/mg DNA) Fig. 4. (a) Kinetics of the binding of varyingconcentrations (0.5--20 nm) of [~H]estradiol to nuclear ER of young(O O) and old ( x x ) rats, and Co)its analysisby Scatchard plot.

decreased from 1.56 p m o l / m g D N A in young to 1.05 p m o l / m g D N A in the old (Table Ib).

Retention time of ER complexes Figure 5 shows the retention time o f [3H]ER complexes in uterine nuclei of young and old rats. In both ages, the amount o f radioactivity bound to nuclei increases up to 1 h. The level o f ER complexes falls remarkably after 1 h in old, but this is main-

119

talned until 2 h in young and drops suddenly thereafter. However, the extent of binding is conspicuously higher in young than in old rats. When the amount of radioactivity bound to salt-resistant nulcear fractions is compared between two ages, it follows the same pattern as in total nuclei. The decrease in the level of specifically bound [3H]ER complexes after 1 h is gradual in young but abrupt in old. While in young more than 60% are retained after 4--6 h, approximately 50% are lost only after 2 h in old.

Binding to DNA-cellulose The binding abifity of the receptor extracted with high salt buffer from the uterine nuclei of young and old rats was studied by DNA cellulose chromatography (Fig. 6). As shown in Fig. 6a, 61% of the receptors isolated from young rats was retained on the affinity resin. About 2/5 of this could be eluted with 0.15 M KCI and the

e - . - - e YOUNG ( T o t a l ) 0.---'0 YOUNG ( S a l t - r e t i a t a n t ) H OLO (Total)

g.O

,.5.0 o

z~3"O o

~'2.0

1-0

-

1

I 2 RETENTION

I 3

I 4 TIME ( h )

t 5

j S

IN N U C L E I

Fig. 5. Retention time of ER complex in total and salt-resistant nuclear fractions of young and old rats.

120

25

A

20

%

0-15 M

15-

I

KCI

0,5 MKCI /

I

~l I

Z

/

I

121

I

/A\

,o

5

0

L 0

I

.........

2

I

4

6

E / U T I O N VOLUME (ml)

100 [] []

Total binding Eluted with 0.15M KCI

[ ] E|uted wit h O-$M KCl

Z Z

\\\

U IL U U.I O.

~n

50

\\\ \\\ \\\ \\\

_ I

.-4 \ \\

%0 0

\\\)o°~ Y OUNG

OLD

Fig. 6. (a) Binding o f the nuclear ER from young (O O) and old ( x x ) rats to a DNA-cellulose column and stepwise elution with T E D G buffer containing first 0.15 M KCI and then 0.5 M KCI. (b) The percentage of specific binding o f nuclear ER from young and old rats to DNA-cellulose. The percentage of total receptors and those eluted with 0.15 M and 0.5 M KCI are presented.

121 remaining 3/5 with 0.5 M KCI. On the other hand, only 37070 of total receptors extracted from old rats bound to the column. About 3/5 of this was eluted with 0.15 M KC1 and the remaining 2/5 with 0.5 M KC1 (Fig. 6b). DISCUSSION Recent studies have demonstrated that both unoccupied and ligand-bound ER are localized in the nuclei of estrogen responsive cells. Furthermore, it has been proposed that both unoccupied and ligand-bound receptors are associated with nuclear components. However, unoccupied receptors are generally easily extracted into cytosol upon homogenization in hypotonic buffers, whereas ligand-bound receptors remain associated with the nuclear pellet unless homogenized in the presence of high salt concentrations. Following injection of estradiol into a rat, the loosely-bound receptors become tightly associated [13] and are not related into the cytosol during the isolation of nuclei. Thus the nuclear preparation is enriched in receptors. When this is extracted with buffer containing high salt (0.5 M KCI), majority of the receptors are released into the nuclear extracts. Different physio-chemical properties of these ER complexes prepared from the uterine nuclei of young and old rats have been compared in the present study. Furthermore, it has been examined whether such difference in structure affects the ligand- and/or DNA-binding properties of nuclear ER. We could not detect any appreciable differences in sucrose density gradient sedimentation profiles and in gel filtration analysis of nuclear ER prepared from the uteri of young and old rats. In both age groups, the two forms of receptors sedimented at 6.8 S and 4.1 S with R s values of 7.2 and 3.3 nm, respectively. Also, the fractional ratio of fast (1.7) and slowly (1.2) sedimenting form of nuclear receptors did not vary in two ages. Similarly, the half life of these receptors showed identical values in young and old. It is likely that ER extracted from the nuclei form complexes with various nuclear components. Probably this complex sediments at 6.8 S and represents a composite of multiple receptor forms. The proportion of this form is higher in old age. Another form of receptor that sediments at 4.1 S perhaps represents the functional form and its level is comparatively greater in young rats. Thomas and Leung [15] demonstrated that different forms of nuclear ER were released during the course of micrococcal nuclease or deoxyribonuclease I digestion of chromatin-bound receptors. These data are consistent with present findings. Similar observation was noted for cytoplasmic receptors by Gesell and Roth [16]. They found all receptors from mature and senescent rats in 7.8 S peak under low salt conditions and in 4 S peak under high salt conditions. Heterogeneity in sizes of uterine ER has been shown by various investigators. We have calculated molecular weight of uterine nuclear ER as 57 K which is equivalent to or slightly less than the values of 60--66 K obtained by SDS-polyacrylamide gel electrophoresis of purified ER [17,18]. This discrepancy may be due to the use of

122 different techniques for molecular weight determination. Alternatively, this may represent the truncated form of ER. It has been reported that in the presence of estradiol, ER undergoes proteolytic cleavage in vivo resulting in the production of truncated form of receptor having lower molecular weight (54--57 K). However, this form is fully active in binding to hormone as well as DNA [19,20]. From the Scatchard analysis of the binding o f 17fl-estradiol to nuclear ER, it is apparent that dissociation constant remains similar in both ages, but the number of binding sites decreases significantly in the old. Chuknyiska et al. [7] also observed no age differences in association or dissociation constant for the nuclear binding reaction of ER. However, they suggested that the inability o f some aged receptors to bind to the full complement of mature nuclear acceptor sites may indicate a qualitative deficiency in the cytosol of old uteri. It is believed that in order to bring about its response, estrogen has to stay in the nuclei of target cells for 4--6 h [21]. Uterine cells contain about 15 000--20 000 receptors in the cytoplasm but only a fraction of this (1000--3000 receptors per cell) is bound to acceptor site and thus retained for long time in the nucleus. These receptors are required for the growth of uterus [10,22]. The ER complexes bound to specific acceptor sites are salt-resistant. The rate o f dissociation of these complexes is considerably slower when bound to chromatin rather than when unbound or free [21]. It is evident from our data that more receptors are retained for longer periods in young nuclei than in old. The duration of nuclear occupancy of ER complexes is correlated with differences in uterine responsiveness to estrogen. Longer duration of estrogen retention in young may be due to greater affinity o f ER complexes to chromatin or due to absence of factors that degrade or inactivate nuclear ER resulting in the translocation of ER from nuclei to cytoplasm. Further binding of nuclear ER complexes to DNA clearly shows that less number of ER complexes is able to bind DNA in old and out of the total bound radioactivity, only 2/5 is tightly bound. This points out that efficiency as well as affinity of ER complexes to bind DNA decreases with age. Despite many similarities in physico-chemical properties of nuclear ER between the uterus of young and old rats, it is likely that there are age-related differences in the conformation of these receptors. It remains to be determined if these differences reflect an impairment in the interaction of nuclear ER with chromosomal sites or specific DNA sequences flanking estrogen responsive genes. However, it is evident from the present findings that the number of estrogen binding sites and the retention time of ER complexes in nuclei and the ability o f these complexes to bind to DNA decrease in old age, albeit the nuclear receptors have many similar physico-chemical properties in the uteri of young and old rats. ACKNOWLEDGEMENTS We wish to thank Prof. M.S. Kanungo and Dr. M.M. Chaturvedi for helpful dis-

123

cussions. The work was supported by grants from the Council of Scientific and Industrial Research, and University Grants Commission, India. REFERENCES 1 2 3

4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22

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