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. REFERENCES 1. 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