A specific glucocorticoid binding macromolecule of rabbit uterine cytosol

BIOCHEMICAL

Vol. 54, No. 2, 1973

A SPECIFIC

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

GLIJCOCORTICOID BINDING MACROMOLECULE OF RABBIT UTERINE CYTOSOL* George

Giannopoulos**

Department

of Experimental Medicine, McGill Royal Victoria Hospital,

Received

July

30,

University Montreal,

and the University Canada.

Clinic,

1973

SUMMARY. A high affinity (Kd=2.7 x 10-l' M at 0') dexamethasone binding macromolecule has been identified in the cytosol fraction of rabbit uteri. Competition studies show high specificity for glucocorticoids since binding of labeled dexamethasone is inhibited by cortisol and corticosterone but not by progesterone, testosterone, or estradiol 178. The binding component has a sedimentation coefficient of 8s and its concentration in uterine cytosol is about 0.2 pmoles per mg protein. Uptake of labeled dexamethasone by isolated uterine nuclei requires the presence of cytosol and is temperature dependent. The KCl-extractable nuclear complex sediments at 4s. Thus the dexamethasone binding components of the rabbit uterus have properties similar to those described for steroid hormone receptors present in target tissues. Specific dexamethasone binding could not be demonstrated in rat uterine cytosol. INTRODUCTION. estrogens,

The uterus

progestins

uterine

growth

a partial

(2),

effect

action

they

event

to the nucleus

where

modif ication in most action

of estrogens

species uterus

has been contains

is a target

for

The present for

and estradiol-17

in rabbit

cortisol

*

Supported

**

Scholar

by Grant

of the Medical

from

Research

@ 1973 by Academic Press, Inc. of reproduction in any form reserved.

This

suggesting

in-

a direct

investigated been

it

cytosol.

used

specific

the Medical Council 600

This

not

for

via

have been

in uteri shown (7).

of several

that If

the rat

the uterus

glucocorticoid

of a dexamethasone

progesterone, specificity

dexamethasone Research

a found

The inter-

macromolecule

binding

of Canada.

complex

(5).

to contain

the presence but

steroid-receptor

receptors

expect

to specific

to be initiated

testosterone

has a high

binding

molecules

has also

for

uterine

the methods

believed

It

demonstrate

therefore

MA-4744

are

specific

(6).

and corticosterone and it

B* However, with

ticoids.

with

one would

studies

of the

have been

proteins

glucocorticoids,

macromolecule

affinity

which

binding

is

Such receptor

(5) .

extensively

specific

receptors. binding

changes

and progesterone studied

and cause

(3,4).

animals

hormones

by translocation

tissues

imbibition

(2).

of steroid

expression

steroid-sensitive

fluid

including

do not promote

to estrogens

in hypophysectomized

physiological

of genetic

of hormones

glucocorticoids

responses

in the uterus followed

a variety

Although

growth

in the action

receptors

for

the estrogen-induced

observed

of glucocorticoids

cytoplasmic

Copyright All rikhts

inhibit

(1).

of long-term is also

An early

organ

and androgens

inhibition

hibitory

is a target

Council

also

has an

testosterone for binding

glucocorcould

of Canada.

not

BIOCHEMICAL

Vol. 54, No. 2, 1973

0.5

1

2

4

3

AND BIOPHYSICAL

5

RESEARCH COMMUNICATIONS

6

1.0

0.5

1.5

2.0

Bound(M.lO'O)

Free(Mxl0')

Fig. 1. A. Concentration-dependence of binding of 3 H-dexamethasone to rabbit uterine cytosol. Aliquots jf cy tosol were incubated for 2 hrs at 0' with increasing concentrations of H-dexamethasone alone or together xiih an 100-fold concentration of non-labeled dexamethasone. 3PDexamethasone binding was measured by a charcoal assay (8). Specific binding of 3Wdexamethasone was calculated by subtracting the amount bound in the presence of loo-fold excess of non-labeled steroid (non-specific binding) from the total binding (in the absence of non-labeled steroid). B. Scatchard plot of 3H-dexamethasone to rabbit uterine cytosol. Data were taken from the results shown in A.

be demonstrated METHODS. mature

in the

Mature

hooded

studies.

Uteri

buffer,

pH 7.4,

cytosol

were

ized

with

measured

by sucrose

of sucrose

by a charcoal

gradient

nuclei

RESULTS.

earlier

When rabbit

of the affinity

binding

The concentration ments

Specific

data

according

sites

with

of binding whether

by adsorption to Scatchard

(9)

a dissociation sites

dexamethasone

601

bound

and methods

of radioactivity by isolated

3.

hormone (Fig.

concentrations on charcoal, lA).

A plot

a single

of 2.7 from

per mg of cytosol is

assay

increasing

was observed

constant

was calculated

0.2 pmoles

to Fig.

indicated

alone

character-

of 3H-dexamethasone

of the free

sites

were

and measurement

with

of the

of 3H-dexamethasone

components

was incubated

of binding

was prepared

Aliquots

The charcoal uptake

being

Tris-HCl

(22 Ci/mmole)

binding

in the legend

cytosol

number

to be approximately To determine

(8).

30 min.

Kg) and

in these

The cytosol

x g for

fractionation,

as described

used

of O.OlM

3H-dexamethasone

and the binding

2.7-3.2

15 days before (w/v)

Specific

wt.

g) were

A).

centrifugation.

and analyzed

of a limited binding

at 224,000

assay

uterine

5 volumes

steroid.

gradient

was studied

in

at 0' with

centrifugation,

of 3H-dexamethasone saturation

2 hrs

(body

200-250

Na2EDTA (Buffer

non-labeled

density

have been described uterine

for

wt.

uterus. rabbits

or adrenalectomized

of the homogenate incubated

or in combination was then

intact

0.0015M

White

(body

and homogenized

containing

by centrifugation

rats

either

minced

of the rat

New Zealand

female

were

were

fraction

female

Long Evans

The rats

used.

cytosol

intact

the

class of high -10 x 10 M (Fig. 1B). results of eight experi-

protein.

to macromolecular

components,

the

Vol. 54, No. 2, 1973

BIOCHEMICAL

3H-Dexamethasone

I 4900

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

c Dexamethasone

2900

W.

+lO-‘M Carllsol

t

it

I

4400

Es,rad,ol

+lO-‘M

2

-17”

2400 t

400

1

300

200

100 I-J

1

1

I

I

1

I

I

0

10

20

30

10

20

30

Fraction Number 7

I

Fig. 2. Sedimentation of bound aH-dexamethasoneon sucrose gradients and competition by various non-labeled steroids for binding of 3H-dexamethasone to rabbit uterine cytosol. Aliquots of the cytosol were incubated for 2 hrs at Oo with 1 x lo-8M 3H-dexamethasonealone or together with 1 x lo-711 of nonlabeled steroid. Sampleswere layered on lo-30% sucrose gradients prepared in Buffer A and centrifuged for 16 hours at 55,000 rpm in a SW56rotor. BSA, bovine serum albumin. cytosol was incubated with 1 x 10m8M3H-dexamethasoneand aliquots were layered on 10-30X sucrose gradients containing Buffer A. A clear peak of 3H-dexamethasone binding was observed (Fig. 2).

The 3H-dexamethasonebinding material had a sedi-

602

Vol. 54, No. 2, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10

20

Fraction

Number

Fig. 3. Effect of uterine cytosol and temperature on the uptake of 3H-dexamethasone of isolated uterine nuclei. Rabbit uteri were homogenized in 0.25M sucrose-O.OlM Tris-O.OOlM MgC12buffer pH 7.4 (Buffer B). The homogenatewas filtered through four layers of cheesecloth and centrifuged at 700 x g for 10 min. The 700 x g supernatant was centrifuged for 30 min at 224,000 x g to prepare the cytosol. The 700 x g pellet was washed 3 times with Buffer B to obtain a washed nuclear pellet. Aliquots of the nuclei were suspended either with Buffer B containing 1 x lo-811 3H-dexamethasoneor with the uterine cytosol which had been incubated with 1 x lo-8M 3H-dexamethasoneat Oo for 2 hrs. The nuclear suspensions were incubated for 30 min at Oo or 250. Following incubation, the nuclei were centrifuged, washed 3 times with buffer, and extracted with Buffer A containing 0.4M KCl. Aliquots of the nuclear extracts were layered on 5-20X sucrose gradients prepared in Buffer A containing 0.4M KC1 and centrifuged for 16 hrs at 55,000 rpm in a SW56rotor. w , nuclei incubated with uterine cytosol at 250; 0-0 ,nuclei incubated with uterine cytosol at 00; bp , nuclei incubated with Buffer B at 250. mentation coefficient

of approximately 8s.

The specificity

of dexamethasone

binding to uterine cytosol and the relative affinity of the binding components for various steroids was examined by sucrose gradient and charcoal adsorption analysis. The stereo-specific nature of the binding sites was inferred from the observations that dexamethasone and cortisol completely inhibited binding of 3Hdexamethasone to the 8s component whereas progesterone, testosterone

and estradiol

178 did not compete for binding sites.

Corticosterone also inhibited binding of 3H-dexamethasonebut it was less effective than dexamethasone and cortisol (Fig. 2). Similar results were obtained by charcoal analysis (data not shown). When isolated uterine nuclei were incubated with buffer containing 3Hdexamethasone, specific uptake of the hormone was not observed. Incubation of the nuclei with 'H-dexamethasone in the presence of the uterine cytosol resulted in significant hormone uptake at 25' but not at 0'. Following extraction of

603

Vol. 54, No. 2, 1973

BIOCHEMICAL

TABLE 1.

Source

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SPECIFIC BINDING OF 3H-DEXAMETHASONE TO RABBIT AND RAT UTERINE CYTOSOL.

of uterine cytosol

Bound 3H-Dexamethasone* (pmoles/mg protein +- SEM)

Rabbit (intact) Rat (intact) Rat (adrenalectomized)

0.19 + 0.03 not detected not detected

*Aliquots of cytosol (5.8-7.9 mg protein per ml) prepared from rabbit or rat uteri were incubated for 2 hrs at Oo with 1 x 10m8M 3H-dexamethasone alone or in combination with 1 x lo-6M non-labeled dexamethasone. Specific binding of 3H-dexamethasone was measured by a charcoal assay (8). The values shown represent the mean of 6 experiments in duplicate or triplicate.

the nuclei

with

on 5-20X

sucrose

sedimenting uterine

nuclei

cytosol

4s (Fig. is

Thus specific dependent

successful.

in cytosol

fractions

(Table 1). Similarly, 3 of H-dexamethasone with

of uteri

corticoid

binding

The concentration binding

reported

for rat

requires molecules

of charcoal

from

of cytosol.

in rat

intact

uterine could

or adrenalectomized

analysis components

liver

did

not

or rat

the presence described receptors

A specific

binding

show asso-

uterine

sites including

and sedimentation It

has also

rabbit

of cytosol. here

is

that

of target role

for

mature

to those

tissues

(5).

and the

rabbit

constant,

similar

to those

liver uptake

glucocorticoid

similar

gluco-

dissociation are

specific

the glucocorticoid

604

uterus

(8)

reported receptors

and

of dexa-

temperature-dependent

Thus the uterine

have properties

in a variety physiological

nuclei

sexually

in fetal

been shown

uterine

from its

analysis

affinity,

in rabbit

coefficient,

receptors

gradient

high

of uteri

binding

component,

and sucrose of specific,

in the cytosol

glucocorticoid

(10).

by isolated

presence

of specific

specificity specific

adsorption

the

macromolecules

of the uterine

steroid adult

by

of 3H-dexamethasone

gradient

macromolecular

to demonstrate

rabbits.

methasone

the presence binding

binding

sucrose

components

shown). The techniques

have been employed

properties

of the extract with

of 3H-dexamethasone

dexamethasone Specific

rats

cytosol (not DISCUSSION.

uptake

and requires

to demonstrate

have not been

ciation

steroid

3).

temperature

attempts

be detected

adult

containing 0.6M KC1 and centrifugation 3 H-dexamethasone was associated

gradients,

near

To date, not

buffer

and binding for

macro-

other

in the rabbit

Vol. 54, No. 2, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

uterus cannot yet be assigned, although it may be speculated that the overall regulation of uterine growth and function is the result of many hormones, including the glucocorticoids, acting in concert. An important function of glucocorticoids may be associated with the onset of parturition. Administration of ACTHor glucocorticoids to immature fetal lambs leads to premature delivery (11) and bilateral

fetal

adrenalectomy prolongs pregnancy (12).

Parturition

induced by

infusion of ACTHor glucocorticoids is associated with a fall in plasma and myometrial concentrations of progesterone, a rise in plasma concentration of free estradiol-17B

and a rise in the concentration

maternal cotyledons and myometrium (13).

of prostaglandin

F2d in

It has also been shown recently

that administration of glucocorticoids accelerate parturition of a direct action of glucocorticoids (14) * The possibility

in the rabbit in the uterus to

promote metabolic changes, such as increased levels of prostaglandins, leading to parturition and the mechanismby which they exert these effects are areas of future

study.

The failure in the present studies to detect specific dexamethasone binding in rat uterine cytosol does not necessarily imply that glucocorticoid receptors are not present in this tissue. It is possible that glucocorticoid receptors in rat uterus are present at very low concentrations

or that they

are very unstable and therefore not detectable with the methods used. A second possibility is that the rat uterus contains receptors with high affinity for corticosterone,

the major glucocorticoid

in the rat;

but not dexamethasone.

Such a corticosterone-specific

binding protein has recently been reported to be present in rat kidney (15). Milgrom and Baulieu (16) have reported that rat uterine cytosol contains a protein which binds cortisol. This protein, however, is not specific for glucocorticoids since it also binds progesterone and appears to be very similar, unlikely

if not identical,

that the rat uterine

to serum transcortin.

Therefore,

it is

protein described by Milgrom and Baulieu (16)

represents a specific glucocorticoid receptor. ACKNOWLEDGEMENT. I wish to thank Dr. S. Solomon for critically manuscript and Ms. A. Dillon for technical

reviewing the

assistance.

REFERENCES (1) (2) (3) (4) (5) (6)

Lerner, L.J., Recent Progr. HormoneRes. 3. 435, 1964. Velardo, J.T., Ann. N.Y. Acad. Sci. 2, 441, 1959. Velardo, J.T., Hisaw, F.L. and Bever, A.T., Endocrinology 59, 165, 1956. Szego, C.M. and Roberts, C., Recent Progr. HormoneRes. 8, 419, 1953. Raspe, G. (ed), Advances in the Biosciences 7, PergamonPress, Vieweg, 1971. Jensen, E.V. and DeSombre, E.R., in Biochemical Actions of Hormones (G. Litwack, ed), Vol II, p. 215, Academic Press 1972.

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