0013-7227/79/1046-1563$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 104, No. 6 Printed in U.S.A.
The Intracellular Distribution of Natural and Synthetic Glucocorticoids in the AtT-20 Cell* FRANK SVECf AND ROBERT W. HARRISON:}: Endocrinology Division, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
ABSTRACT. Both natural and synthetic glucocorticoids suppress ACTH production by AtT-20 mouse pituitary cells. However, the degree of suppression is not clearly related to cytosol receptor affinity. These studies were done to test whether the synthetic and natural glucocorticoids might differ in the efficiency with which they caused nuclear translocation of the cytosol receptor. If so, this parameter as well as receptor affinity might contribute importantly to steroidal potency. To accomplish these studies, a rapid method of assaying glucocorticoid binding to whole cells and to isolated nuclei was developed. Whole cell and nuclear binding of triamcinolone acetonide (9a-fluoro-11/8,16a, 17,21-tetrahydroxypregna-1,4diene-3,20-dione cyclic 16,17-acetal with acetone), dexamethasone, and corticosterone were measured after incubation of the steroids with intact cells at 25 C. Uptake of all three steroids
I
T IS GENERALLY assumed that glucocorticoids influence target cell functions through interactions with nuclear constituents which lead to altered gene expression (1, 2). Because of this assumption, we and others have studied factors which influence the specificity and extent of glucocorticoid receptor-nuclear interactions (36). We have found the mouse pituitary AtT-20 cell line to be advantageous as a model for elucidating the mechanism of glucocorticoid action since these cells synthesize ACTH and contain a high affinity cytosol glucocorticoid receptor (7). Glucocorticoids inhibit the production of ACTH (8), and it is postulated that steroid receptornuclear interactions are instrumental in mediating this effect. In quantitating the efficacy of a series of potent glucocorticoid agonists in suppressing ACTH production, it was shown that dexamethasone and triamcinolone acetonide1 were both significantly more effective than Received May 1, 1978. * This work was supported by NCI Grant CA-19907 and Grant AM17026 from the Vanderbilt University Diabetes/Endocrinology Research Center. t Supported by NIH Postdoctoral Fellowship AM-05958. X Investigator for the Howard Hughes Medical Institute. To whom requests for reprints should be addressed. 1 The following trivial name is used: triamcinolone acetonide, 9afluoro-11/8,16a, 17,21 - tetrahydroxypregna- l,4-diene-3,20-dione cyclic 16,17-acetal with acetone.
reached apparent equilibrium by 1 h at 25 C. A Scatchard analysis of binding over a concentration range of 0.1-50 nM indicated approximately 75,000 binding sites/cell for each glucocorticoid. The apparent dissociation constants (K _i
10 I MINUTES
FIG. 1. The effect of incubation in MAT buffer on cellular binding. AtT-20 cells (5 x 10G/ml) were labeled at 25 C for 1 h with 11.5 nM [3H]triamcinolone acetonide, 12.8 nM [:)H]dexamethasone, or 28.8 nM [3H]corticosterone. Nonspecific binding was estimated using a similar incubation containing a 1000-fold excess of unlabeled glucocorticoid. All subsequent procedures were done at 0 C. Aliquots of the suspension (0.2 ml) were placed in conical tubes containing 12 ml MAT buffer and allowed to stand for the indicated time before centrifugation at 2000 rpm for 5 min. Only displaceable binding is shown. • — • , Triamcinolone acetonide; O- -O, dexamethasone; A- -A, corticosterone.
1565
TABLE 1
A. The effect of MgCl2 concentration on the recovery of [l4C]thymidine" cpm/10(i Cells (±SD) 5496 ±247 4222 ±276 4358 ± 1164 4095 ± 132
Total applied 1.5 mM MgCl2 3.0 mM MgCl2 6.0 mM MgCl2
B. Comparison of [14C]thymidine recovery in 3.0 mM MgCl2-0.05% Triton X-100 and Tris-saline bovine serum albumin* 0.05% Triton X-1003.0 mM MgCl2
Tris-saline bovine serum albumin
359 ± 53
345 ± 71
i
cpm/106 Cells (±SD)
l4
" Cells which were grown in 20 nCi/ml [ C]thymidine for 4 h at 37 C were placed in solutions of 0.05% Triton X-100 and the indicated concentrations of MgCl2. After centrifugation, the pellets were analyzed for trichloroacetic acid-insoluble [l4C]thymidine, as outlined in Materials and Methods. h Cells which were grown in 2.5 nCi/ml [14C]thymidine for 3 h at 37 C were placed in either Tris-saline-bovine serum albumin or the 0.05% Triton X-100-3 mM MgCl2 solution. After centrifugation, the pellet was analyzed for trichloroacetic acid-insoluble [14C]thymidine, as outlined in Materials and Methods.
at 5 or 7 min. Cell breakage and loss of radioactivity was minimal if the cells were placed in Tris saline at 0 C. A similar analysis showed that the nuclear pellet obtained from cells incubated with labeled triamcinolone acetonide, dexamethasone, or corticosterone at 0 C was devoid of radioactivity, suggesting that this procedure could be employed as a rapid accurate assessment of steroid receptor-nuclear uptake. The percentage recovery of applied DNA was assessed for the whole cell assay procedure and the nuclear isolation procedure using cells labeled with [14C]thymidine. Nuclear material was lost during both procedures. The recovery of DNA was approximately 80% in each instance and the concentration of MgCl2 did not affect recovery (Table 1A). Since the recovery of DNA was the same for both the whole cell and the nuclear assay techniques (Table IB), direct comparisons between the assays are valid. Whole cell and nuclear uptake of glucocorticoids Glucocorticoids bound readily to intact AtT-20 cells. As seen in Fig. 2, the uptakes of dexamethasone and triamcinolone acetonide reached their equilibrium values after approximately 45 min at 25 C. The rate of corticosterone uptake was faster and reached its equilibrium in 15-30 min. As is also shown in Fig. 2, specific nuclear binding attained a stable value in a period of time similar to that of the whole cell uptake. Therefore, to ensure that equilibrium conditions were reached in subsequent experiments, incubations were carried out for 1 h.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 16:56 For personal use only. No other uses without permission. . All rights reserved.
SVEC AND HARRISON
1566
12-t
The experimental strategy was to assess whole cell and nuclear binding of the three glucocorticoids over a wide range of physiologically relevant concentrations. The results of these experiments are shown in Fig. 3. The concentrations employed were sufficient to approach saturation of both nuclear and whole cell binding o
DEXAMETHASONE
8
Endo • 1979 Vol.104 • No 6
DEXAMETHASONE
10-
.08-]
8-
.06-
6-
.04-
4-
.02-
2-
o-
0
J
o ?
0
12 1
2
4
8 12
4
TRIAMCINOLONE ACETONIDE D
4-
LU O
OJ
0
30
60
90
120
TRIAMCINOLONE ACETONIDE
LJJ
§ O
a: LU
40J f 0 2 4 6 CONCENTRATION OF STEROID
o
0
60
30
90
120
90
120
00 CORTICOSTERONE A
-A—A-
Vol. 104, No. 6 Printed in U.S.A.
The Intracellular Distribution of Natural and Synthetic Glucocorticoids in the AtT-20 Cell* FRANK SVECf AND ROBERT W. HARRISON:}: Endocrinology Division, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
ABSTRACT. Both natural and synthetic glucocorticoids suppress ACTH production by AtT-20 mouse pituitary cells. However, the degree of suppression is not clearly related to cytosol receptor affinity. These studies were done to test whether the synthetic and natural glucocorticoids might differ in the efficiency with which they caused nuclear translocation of the cytosol receptor. If so, this parameter as well as receptor affinity might contribute importantly to steroidal potency. To accomplish these studies, a rapid method of assaying glucocorticoid binding to whole cells and to isolated nuclei was developed. Whole cell and nuclear binding of triamcinolone acetonide (9a-fluoro-11/8,16a, 17,21-tetrahydroxypregna-1,4diene-3,20-dione cyclic 16,17-acetal with acetone), dexamethasone, and corticosterone were measured after incubation of the steroids with intact cells at 25 C. Uptake of all three steroids
I
T IS GENERALLY assumed that glucocorticoids influence target cell functions through interactions with nuclear constituents which lead to altered gene expression (1, 2). Because of this assumption, we and others have studied factors which influence the specificity and extent of glucocorticoid receptor-nuclear interactions (36). We have found the mouse pituitary AtT-20 cell line to be advantageous as a model for elucidating the mechanism of glucocorticoid action since these cells synthesize ACTH and contain a high affinity cytosol glucocorticoid receptor (7). Glucocorticoids inhibit the production of ACTH (8), and it is postulated that steroid receptornuclear interactions are instrumental in mediating this effect. In quantitating the efficacy of a series of potent glucocorticoid agonists in suppressing ACTH production, it was shown that dexamethasone and triamcinolone acetonide1 were both significantly more effective than Received May 1, 1978. * This work was supported by NCI Grant CA-19907 and Grant AM17026 from the Vanderbilt University Diabetes/Endocrinology Research Center. t Supported by NIH Postdoctoral Fellowship AM-05958. X Investigator for the Howard Hughes Medical Institute. To whom requests for reprints should be addressed. 1 The following trivial name is used: triamcinolone acetonide, 9afluoro-11/8,16a, 17,21 - tetrahydroxypregna- l,4-diene-3,20-dione cyclic 16,17-acetal with acetone.
reached apparent equilibrium by 1 h at 25 C. A Scatchard analysis of binding over a concentration range of 0.1-50 nM indicated approximately 75,000 binding sites/cell for each glucocorticoid. The apparent dissociation constants (K _i
10 I MINUTES
FIG. 1. The effect of incubation in MAT buffer on cellular binding. AtT-20 cells (5 x 10G/ml) were labeled at 25 C for 1 h with 11.5 nM [3H]triamcinolone acetonide, 12.8 nM [:)H]dexamethasone, or 28.8 nM [3H]corticosterone. Nonspecific binding was estimated using a similar incubation containing a 1000-fold excess of unlabeled glucocorticoid. All subsequent procedures were done at 0 C. Aliquots of the suspension (0.2 ml) were placed in conical tubes containing 12 ml MAT buffer and allowed to stand for the indicated time before centrifugation at 2000 rpm for 5 min. Only displaceable binding is shown. • — • , Triamcinolone acetonide; O- -O, dexamethasone; A- -A, corticosterone.
1565
TABLE 1
A. The effect of MgCl2 concentration on the recovery of [l4C]thymidine" cpm/10(i Cells (±SD) 5496 ±247 4222 ±276 4358 ± 1164 4095 ± 132
Total applied 1.5 mM MgCl2 3.0 mM MgCl2 6.0 mM MgCl2
B. Comparison of [14C]thymidine recovery in 3.0 mM MgCl2-0.05% Triton X-100 and Tris-saline bovine serum albumin* 0.05% Triton X-1003.0 mM MgCl2
Tris-saline bovine serum albumin
359 ± 53
345 ± 71
i
cpm/106 Cells (±SD)
l4
" Cells which were grown in 20 nCi/ml [ C]thymidine for 4 h at 37 C were placed in solutions of 0.05% Triton X-100 and the indicated concentrations of MgCl2. After centrifugation, the pellets were analyzed for trichloroacetic acid-insoluble [l4C]thymidine, as outlined in Materials and Methods. h Cells which were grown in 2.5 nCi/ml [14C]thymidine for 3 h at 37 C were placed in either Tris-saline-bovine serum albumin or the 0.05% Triton X-100-3 mM MgCl2 solution. After centrifugation, the pellet was analyzed for trichloroacetic acid-insoluble [14C]thymidine, as outlined in Materials and Methods.
at 5 or 7 min. Cell breakage and loss of radioactivity was minimal if the cells were placed in Tris saline at 0 C. A similar analysis showed that the nuclear pellet obtained from cells incubated with labeled triamcinolone acetonide, dexamethasone, or corticosterone at 0 C was devoid of radioactivity, suggesting that this procedure could be employed as a rapid accurate assessment of steroid receptor-nuclear uptake. The percentage recovery of applied DNA was assessed for the whole cell assay procedure and the nuclear isolation procedure using cells labeled with [14C]thymidine. Nuclear material was lost during both procedures. The recovery of DNA was approximately 80% in each instance and the concentration of MgCl2 did not affect recovery (Table 1A). Since the recovery of DNA was the same for both the whole cell and the nuclear assay techniques (Table IB), direct comparisons between the assays are valid. Whole cell and nuclear uptake of glucocorticoids Glucocorticoids bound readily to intact AtT-20 cells. As seen in Fig. 2, the uptakes of dexamethasone and triamcinolone acetonide reached their equilibrium values after approximately 45 min at 25 C. The rate of corticosterone uptake was faster and reached its equilibrium in 15-30 min. As is also shown in Fig. 2, specific nuclear binding attained a stable value in a period of time similar to that of the whole cell uptake. Therefore, to ensure that equilibrium conditions were reached in subsequent experiments, incubations were carried out for 1 h.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 16:56 For personal use only. No other uses without permission. . All rights reserved.
SVEC AND HARRISON
1566
12-t
The experimental strategy was to assess whole cell and nuclear binding of the three glucocorticoids over a wide range of physiologically relevant concentrations. The results of these experiments are shown in Fig. 3. The concentrations employed were sufficient to approach saturation of both nuclear and whole cell binding o
DEXAMETHASONE
8
Endo • 1979 Vol.104 • No 6
DEXAMETHASONE
10-
.08-]
8-
.06-
6-
.04-
4-
.02-
2-
o-
0
J
o ?
0
12 1
2
4
8 12
4
TRIAMCINOLONE ACETONIDE D
4-
LU O
OJ
0
30
60
90
120
TRIAMCINOLONE ACETONIDE
LJJ
§ O
a: LU
40J f 0 2 4 6 CONCENTRATION OF STEROID
o
0
60
30
90
120
90
120
00 CORTICOSTERONE A
-A—A-
