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Estrogen and glucocorticoid receptors in adult canine articular cartilage.

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568
ESTROGEN AND GLUCOCORTICOID RECEPTORS IN
ADULT CANINE ARTICULAR CARTILAGE
PETER C. M. YOUNG and MICHAEL T. STACK
The cytoplasmic and crude nuclear fractions of
adult mongrel dog articular cartilage contained estradi01- and dexamethasone-binding components which had
properties of physiologic steroid receptors. The equilibrium dissociation constants averaged 0.37 nM for estradiol and 2.27 nM for dexamethasone. The concentrations of estrogen receptors ranged from below 6 to 101
f m o l h g protein in the cytosols and from below 2.8 to
17.5 fmol/pg DNA in the nuclear fractions. Glucocorticoid receptors were detected in only 4 of 13 cytosols
(range: 61.2-132 fmolhg protein), whereas 10 of 13
nuclear fractions contained 0.8 to 46.8 femtomoles of the
receptors for each microgram of DNA. There appeared
to be no marked difference between the contents of
either steroid receptor in female or male dog cartilage.
No receptors were detected for androgen and progesterone.
Cartilage has recently been found to bear cellular receptors for both insulin and glucocorticoids ( I ,2).
Whether articular cartilage is a target organ for estrogen, which is known to increase the mechanical
From the Department of Obstetrics and Gynecology and
the Department of Medicine, Division of Rheumatology. Indiana
University School of Medicine, Indianapolis. I N .
Supported in part by Grant SO7 RR 5371 from the U.S.
Public Health Service and Grant AM 20582 from the National
Institutes of Health.
Peter C. M. Young, PhD: Associate Professor of Obstetrics
and Gynecology. Indiana University School of Medicine: Michael
T. Stack. PhD, MD: Assistant Professor of Medicine. Indiana
University School of Medicine. and Research Fellow. Arthritis
Foundation.
Address reprint requests to Peter C. M. Young. PhD.
Indiana University School of Medicine, Dept. of Obstetrics and
Gynecology. I100 W. Michigan St.. Indianapolis. I N 46123.
Submitted for publication June 5 . 1981; accepted in revised
form November 3. 1981.
Arthritis and Rheumatism, Vol. 25, No. 5 (May 1982)
strength of the cartilage growth plate in rats (3). is not
known. Studies in vivo have shown chondrocyte hypertrophy, decreased )'SO4 incorporation, and decreased turnover of proteoglycan in response to estrogen administration (4.5). The mechanism of estrogen's
action on chondrocytes is unknown; blockade of somatomedin effects has been postulated as a mechanism ( 4 3 ) . Srudies in vitro have yielded conflicting
results about "SO4 uptake (4). We have previously
demonstrated that changes of cartilage matrix in vitro.
especially enhancement of hyaluronic acid synthesis,
are mediated by the dibutyryl analog of cyclic adenosine monophosphate (6). A recent study of mouse skin
revealed enhanced hyaluronic acid synthesis in response to estrogen: the response was proportional to
the number of estrogen receptors present in the male
mouse skin used (7).
Other studies have revealed that testosterone
and cortisol also affect cartilage in vivo by enhancing
hyaluronic acid synthesis and depressing 35S04incorporation in vivo. respectively; the mechanisms are
under investigation (4). This study was designed to
determine if receptors for estrogen, progestin. androgen, and cortisol exist in adult canine cartilage, and, if
so, the relative number and cellular compartmentalization of those found.
MATERIALS AND METHODS
Buffers. Two buffers were used in this study. Buffer
A was 3.2 ,,,M ~~j~ buffer base, 1.9 m~ ~ , .buffer-Hcl,
i ~
5
d l HEPES, I .5 mM ethylenediaminetetraacetate(EDTA),
0.5 mM dithiothreitol (D?T), I5 mM sucrose9
NaN37
pH 7.6 at 4°C. 7.4 at 25°C. Buffer B was buffer A that
contained 10% glycerol.
Tissues. Cartilage was collected in iced buffer A from
ESTROGEN AND GLUCOCORTICOID RECEPTORS
both knee joints of large (30-50 kg) adult mongrel dogs, as
previously described (6). The limbs of these animals were
not examined radiologically to determine epiphyseal closure;
however, all the dogs obtained from this breeder that were
examined by x-ray in the past had closed epiphyses. All the
cartilage was grossly normal with smooth, glistening cartilage surface. None of the tissues revealed any fibrillation,
erosions, or osteophytes. Histologic examination was not
performed. The slices from both knees of each animal were
pooled (about 500 mg/dog), but the tissue from each animal
was analyzed separately unless mentioned otherwise. Whenever possible, the tissue was washed in buffer A, blotted,
weighed, and processed immediately. On some occasions,
the tissue was frozen in liquid N2 and stored at -70°C to be
used within 2 weeks of freezing.
Measurement of cytoplasmic and nuclear steroid receptors. The tissue was homogenized in 6 volumes of buffer
A with a Polytron (% speed, 4 bursts of 15 seconds). A
1,OOOg crude nuclear pellet and a 100,OOOg supernatant
(cytosol) were prepared from the homogenate (8,9). The
washed pellet was resuspended in 4 volumes of buffer A and
filtered through 3 layers of cheesecloth to yield the crude
nuclear fraction. Binding of steroids to cytoplasmic receptors was studied by incubating equal portions of the cytosol
with increasing concentrations (0.15-20 nM) of the labeled
hormone in the absence and presence of 300-fold molar
excess of an appropriate competing hormone for 16 hours at
4°C. The final volume was adjusted to 200 pI with buffer A.
At the end of the incubatidn, bound and unbound 'Hestradiol (90 Ci/mmol) were sgparated by dextran-coated
charcoal, as previously described (8). In a similar fashion,
bound and unbound 'H-progesterone (1 13.9 CUmmol) and
'H-methyltrienolone (55.5 Ci/mmol) were assayed. Bound
'H-dexamethasone (47.5 Ci/mmol) was measured by a similar charcoal assay, modified in the following manner: after
200 p1 of 60% glycerol in buffer A was added to the
incubation mixtures and mixed, 500 pl of the charcoal
suspension that contained 30% glycerol was added, the tubes
were vortexed, left standing for 15 minutes with mixing at 5minute intervals, and centrifuged for 10 minutes at 1,OOOg to
sediment the charcoal. Portions of the supernatant fractions
were mixed with 10 ml of Eastman Ready-to-Use I11 for
counting. The binding data were analyzed by the method of
Scatchard (10). In some experiments, the washed nuclear
pellet was extracted with 3 volumes of 0.4M potassium
chloride in buffer A for 2 hours at 4°C. The KCI extract was
diluted 1 :2 with buffer A and the binding of 3H-estradiol and
3H-dexamethasone by the extract was studied as described
for the cytosol.
Nuclear uptake of steroids was measured by incubating 120 pl of the nuclear suspension with 20 nM of the
labeled hormone in the absence and presence of 6 pM of an
appropriate competing hormone. All incubations were done
in triplicate and were carried out at 4°C for 18 hours. In some
of the studies of estrogen uptake, an additional, identical set
of incubations was prepared. After 15 hours of incubation at
4"C, this set was exposed to 28°C for 3 hours and then
immediately chilled in an ice bath for 15 minutes. At the end
of this period, I ml of ice-cold buffer B was added to all the
tubes. After mixing, the tubes were centrifuged at I,000g for
10 minutes. The pellets obtained were washed 2 more times
569
with 1.5 ml buffer B. The washed pellets were broken up by
adding 100 pI of water to each tube and vortexing vigorously
before extraction with 900 pI of ethanol. The extraction was
allowed to proceed for 30 minutes with vigorous mixing at
10-minute intervals. After centrifugation, 800-pl portions of
the supernatant fractions were mixed with 10 ml of scintillation fluid and counted.
In the experiments in which the hormone specificity
of the cytoplasmic and nuclear receptors was studied, 50fold molar excesses of various unlabeled hormones were
tested for their ability to compete with 20 nM of labeled
estradiol or dexamethasone for binding. The methodologies
used were the same as those described above.
Measurement of steroid receptors in chondrocyte suspensions. Fresh cartilage slices from a single female adult
dog were cultured in Ham's F12 medium containing 10%
fetal calf serum, penicillin, streptomycin, and 0.075% (w/v)
collagenase (Worthington CLS I1 125-250 units/mg, Freehold, NJ), as previously described (11). After 22 hours
incubation at 37°C under 95% C02:5% air on a rocking
platform, the cells were harvested by centrifugation at 600g
after filtration of the suspension through two 2-ply gauze
pads. The cells were washed 2 times with phosphate buffered
saline, pH 7.0, and collected each time by centrifugation.
Viability by trypan blue exclusion was determined to be
90%, with a yield of 8 x lo6 cells/400 mg pooled knee cartilage. The washed cells were resuspended in RPMI media
(Gibco) at a final cell concentration of 1.33x lo6 cells/ml.
Estradiol and glucocorticoid receptor assays were performed according to the procedure of Konior Yarbro et al
(12), with minor modifications (13) and changes as noted
below. When 3H-dexamethasone was used as the ligand,
300-fold molar excess of unlabeled triamcinolone acetonide
was added to the incubations as the competing hormone. In
the estradiol receptor assay, 300-fold unlabeled diethylstilbestrol was used to compete with 'H-estradiol for binding.
Estradiol and dexamethasone binding sites for each cell and
their respective equilibrium dissociation constants were determined by analysis of the binding data according to the
method of Scatchard (10).
Protein and DNA determinations. Protein concentration in the cytosols was determined by the method of
Bradford (14), and DNA concentration in the nuclear fractions was measured by the diphenylamine method (15).
RESULTS
Figures 1 and 2 show that the cytoplasmic and
nuclear fractions of canine articular cartilage contain
estrogen- a n d glucocorticoid-binding components that
interact with 3H-estradiol a n d 3H-dexamethasone, respectively, with high affinities and limited capacities.
In t h e cytosols studied, the equilibrium dissociation
constants (Kd's) for estradiol ranged from 0.05-0.83
nM with an average of 0.37 nM (n = S), a n d 1.25-3.45
nM for dexamethasone with an average of 2.27 n M
(n = 4). In the 2 KCI extracts of nuclear fractions
s h o w n here, t h e Kd w a s 0.55 nM for estradiol, and 1.34
nM for dexamethasone.
YOUNG AND STACK
570
0015
\,
0 03
\*
Table 1. Percent inhibition of 'H-estradiol binding to cytosol and
nuclear fractions of canine articular cartilage*
~~
0 010
0 00:
0
0 02
1
.
2
0 0'
0002
0002
0004
0004
Percent inhibitiont
= 0.55 nM
Kd=O23nM
C
0'01
0'02
Competing steroid
Cytosol
Nuclear fraction
Dieth ylstilbestrol
Estradiol
Estrone
Estradiol- 17u
Estriol
Dexamethasone
Cortisol
Progesterone
Testosterone
110.2
100.0
51.6
51.3
28.5
0.1
0
0
0.2
106.5
100.0
64.0
49.4
25.7
0
0
0
0
Figure 1. Scatchard plot of 'H-estradiol binding by cytosol (A) and
0.4M KCI extract of the crude nuclear pellet (B) of adult mongrel
dog articular cartilage. Competing hormone was 300-fold excess of
diethylstilbestrol. Kd = equilibrium dissociation constant.
* Cytosol or nuclear fraction was incubated with 20 nM 'H-estradiol
in the absence and presence of 1 p M unlabeled hormone for 16 hours
at 4°C. Bound hormone was measured as described in Materials and
Methods. Inhibition of 'H-estradiol binding by 1 pM unlabeled
estradiol was taken as 100%.
'r The percentages of inhibition shown are averages of duplicate
determinations.
The cytoplasmic and nuclear steroid-binding
components appeared to be hormone specific. At
concentrations equal to 50 times that of labeled estradiol and dexamethasone, the order of affinities of
various competing hormones for the binding components was: diethylstilbestrol>estradiol>estrone>
estradiol-l7a>estriol for the estrogen-binding components (Table 1); and triamcinolone acetonide>dexamethasone >cortisol >corticosterone > promegestone
(R5020)>medrox yprogesterone acetate >progest erone
for the glucocorticoid-binding components (Table 2).
Progesterone, testosterone, cortisol, and dexametha-
sone did not compete with 3H-estradiol for binding
(Table 1). Estradiol, estradiol-I7a, and testosterone
had virtually no affinity for the glucocorticoid-binding
components (Table 2).
The cytoplasmic and nuclear concentrations of
estradiol- and dexamethasone-binding components
were determined in a number of canine articular
cartilage samples. Results in Tables 3 and 4 suggest
that there was no marked difference between the
contents of estradiol- or glucocorticoid-binding components in fresh and frozen cartilages irrespective of
whether the dogs were female (Table 3 ) or male (Table
Bound [nM)
Bound [nM)
B
A
0015
0 010
0 005
0
0015
\=
-
125nM
OOlC
c
001
Bound (nM)
\
002
0 00:
Table 2. Percent inhibition of 'H-dexamethasone binding to
cytosol and nuclear fractions of canine articular cartilage*
1
001
Percent inhibitiont
Kd = 1 34 nM
Competing steroid
Cytosol
Nuclear fraction
003
Triamcinolone acetonide
Dexamethasone
Cortisol
Corticosterone
Promegestone (R5020)
Medroxyprogesterone acetate
Progesterone
Estradiol
Estradiol- 17u
Testosterone
121.2
100.0
88.0
54.0
47.9
40.5
12.6
0
0
0
117. I
100.0
92.3
66.4
40. I
29.6
10.0
0
0
0
002
Bound (nM)
Figure 2. Scatchard plot of 'H-dexamethasone binding by cytosol
(A) and 0.4M KCI extract of the crude nuclear pellet (B) of adult
mongrel dog articular cartilage. Competing hormone was 300-fold
molar excess of cortisol. Kd = equilibrium dissociation constant.
* Cytosol or nuclear fraction was incubated with 20 nM 'H-dexamethasone in the absence and presence of 1 pM unlabeled hormone
for 16 hours at 4°C. Bound hormone was measured as described in
Materials and Methods. Inhibition of 'H-dexamethasone binding by
1 p M unlabeled dexamethasone was taken as 100%.
t The percentages of inhibition shown are averages of duplicate
determinations.
ESTROGEN AND GLUCOCORTICOID RECEPTORS
57 1
Table 3. Cytoplasmic and nuclear concentrations of estrogen- and glucocorticoid-binding
components in female dog articular cartilage*
Estrogen-binding components
Number
of
samples
Fresh
1
1
1
1
Frozen
5
6
3
1
Glucocorticoid-binding components
Cytosol
(fmoV
mg protein)
Nuclear
(fmol/
cLg DNA)
Cytosol
(fmoli
mg protein)
Nuclear
(fmol/
cLg DNA)
Not done
Not detected
10.5
3.0
5.4
4.4
3.6
Not done
Not detected
Not detected
Not detected
Not detected
33.5
0.8
Not done
Not detected
6.0
19.3
11.2
8.1 (8.l)t
Not detected
2.8
11.8 (17.5)t
Not detected
Not detected
83.2
Not detected
13.9
Not detected
6.8
46.8
7.5
* The incubations contained either 20 nM 3H-estradiol with and without 6 p M diethylstilbestrol, or 20
nM 3H-dexamethasone with and without 6 K M cortisol.
t Nuclear exchange assays in which the last 3 hours of incubation were performed at 28°C.
4). The cytoplasmic and nuclear concentrations of
estradiol-binding components are similar in female and
male dog articular cartilages. Cytoplasmic dexamethasone-binding components could be detected in only 1
of 7 samples of female dog cartilages (Table 3 ) , whereas 3 of 6 cytosols from male dog cartilages contained
comparatively higher concentrations of these receptors (Table 4). Five of 7 nuclear fractions from female
dog cartilages showed specific uptake of 3H-dexamethasone, although the binding activity was considerably lower in 2 of the preparations (Table 3 ) . In
contrast, 5 of 6 nuclear fractions obtained from male
dog cartilages contained 16.0 to 36.9 fmol
moles) glucocorticoid-binding components per microgram of DNA (Table 4).
Nuclear uptake of 3H-estradiol at 4°C and 3Hestradiol exchange at 28°C was measured simultaneously in 2 nuclear fractions each of female (Table 3 )
and male (Table 4) dog cartilages. Only 1 of the 4
preparations showed substantial exchange in nuclear
estradiol binding, suggesting that most of the nuclear
estradiol-binding sites of dog cartilage were unoccupied in vivo.
No specific binding was detected in studies of 4
dogs in either the cytosol or nuclear fractions when
labeled methyltrienolone (R1881), medroxyprogesterone acetate, or promegestone (R5020) was used as the
ligand (data not shown). In the incubations in which
3H-R1881 was used, 500-fold molar excess of unlabeled triamcinolone acetonide was added to eliminate
binding of R1881 to progesterone receptors. Since
medroxyprogesterone acetate and R5020 are known to
interact with glucocorticoid and androgen receptors,
100-fold molar excess of unlabeled dexamethasone
and 5a-dihydrotestosterone (DHT) were added when
either of these 2 labeled progestins was used as the
ligand .
In an attempt to exclude artifactual results of
estradiol- and dexamethasone-binding caused by the
possible presence of matrix proteins in the cytosol and
nuclear fraction of the cartilages, 1 experiment was
performed in which a chondrocyte suspension was
prepared following digestion of the canine articular
cartilage with collagenase. From the results of this
experiment, the chondrocytes were found to contain
2,982 estradiol-binding sites per cell with Kd = 0.13
nM, and 6,822 dexamethasone-binding sites per cell
with Kd = 1.13 a.
DISCUSSION
The data presented document, for the first time,
the presence of estrogen and glucocorticoid receptors
in adult canine articular cartilage. We were unable to
detect androgen and progesterone receptors in this
tissue. The presence of glucocorticoid receptors in
embryonic chick growth cartilage has been described
(16), and there is preliminary evidence to show that
macromolecules with some binding characteristics
similar to those of glucocorticoid receptors were also
present in cultured rabbit articular chondrocytes (2).
Interpretation of this latter finding is complicated by
the potential of chondrocytes to dedifferentiate in
YOUNG AND STACK
572
Table 4. Cytoplasmic and nuclear concentrations of estrogen- and glucocorticoid-binding
components in male dog articular cartilage*
Estrogen-binding components
Glucocorticoid-binding components
Cytosol
(fmoV
mg protein)
Nuclear
(fmoU
I.% DNA)
Cytosol
(fmol/
mg protein)
Nuclear
(fmoV
pg DNA)
1
1
9.7
10.8
27.4
9.8
Not detected
Not done
Not detected
116.7
Not detected
29.4
36.9
Not detected
Frozen
1
5
5
Not detected
76.8
101.0
Not detected
8.5 (8.9)t
6.2 (6.2)t
132.0
Not detected
61.2
15.0
16.0
16.1
Number
of
samples
Fresh
1
* The incubations contained either 20 nM 3H-estradiolwith and without 6 jN diethylstilbestrol, or 20
nM 3H-dexamethasone with and without 6 p M cortisol.
t Nuclear exchange assays in which the last 3 hours of incubation were performed at 28°C.
culture to fibroblastic cells, which are known to contain glucocorticoid receptors (17).
To our knowledge, estrogen receptors have not
been demonstrated in articular cartilage, and at the
time of writing, one unsuccessful attempt to detect
specific estrogen and glucocorticoid binding in epiphyseal tissue of young dogs has been reported in abstract
form (18).
These cytoplasmic and nuclear estrogen- and
glucocorticoid-binding components have the binding
characteristics of typical steroid receptors, with high
affinities, limited capacities, and high specificity for
their respective steroid ligands. The relative affinities
of various steroid hormones and diethylstilbestrol for
these binding components reflect, in general, their
biologic potencies in estrogenic or glucocorticoid activity. At the concentrations tested, the 4 classes of
steroid hormones did not cross-react significantly with
these binding components, with the notable exception
of the progestins, which are known to interact with
glucocorticoid receptors (17,19,20). Because the number of dogs used in this preliminary study was small
and the hormonal status of these animals was not
known, it was not possible to determine whether or
not the contents of estrogen- and glucocorticoid-binding components in the articular cartilage were related
to the sex of the dogs.
In 1 female dog, isolated cartilage cells were
shown to possess binding components for both estrogen and glucocorticoid with affinities equal to those
found in whole cartilage extracts. This result validated
the use of whole-cartilage extracts despite the predominance of matrix proteins, which were not expected to
possess specific receptors for these steroids.
The nuclear receptors for both estrogen and
glucocorticoid are extractable by 0.4M KC1 and displayed comparable affinities for their respective hormone ligands. Unlike the estrogen receptors of rat
uterus (21), the nuclear estrogen receptors of dog
cartilage seemed mostly unoccupied by the hormone.
Similar observations have been made in a human
breast cancer cell line (22), in human endometrium
(23), and in human myometrium (24). Under our
experimental conditions, substantial exchange in nuclear estradiol binding was observed in only 1 of 4
crude nuclear preparations after 3 hours of incubation
at 28°C (Tables 3 and 4). We have not investigated
whether exchange was complete under these conditions, and attempts to perform the nuclear exchange
assays at higher temperatures resulted in inactivation
of the estrogen-binding components (results not
shown).
Future investigations will be aimed at correlation of the receptor presence with hormonal effects in
the articular cartilage, in vitro, where glycosaminoglycan metabolism can be monitored.
ACKNOWLEDGMENTS
We thank Mr. Michael Kinch for excellent technical
assistance, and Mrs. Willa Ray and Mrs. Roberta Fehrman
for excellent secretarial assistance.
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