0013-7227/79/1056-1281$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 6 Printed in U.S.A.
In Vitro and In Vivo Binding of Progestins to the Androgen Receptor of Mouse Kidney: Correlation with Biological Activities* TERRY R. BROWN,f LESLIE BULLOCKS AND C. WAYNE BARDIN Departments of Medicine and Comparative Medicine, The Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033
ABSTRACT. Progestins possess androgenic, antiandrogenic, and synandrogenic activities on androgen-responsive tissues. Several recent studies suggest that some or all of these actions may be initiated in mouse kidney via progestin interaction with the androgen receptor. All of the progestins studied, including medroxyprogesterone acetate [6a-methyl-17a-hydroxy-pregn-4ene-3,20-dione acetate (MPA)], cyproterone acetate [6-chloro17a-hydroxy-la,2a-methylene-pregna-4,6-diene-3,20-dione acetate (CPA)], 6a-methylprogesterone (6MP), and progesterone (P), effectively competed with [3H]testosterone ([3H]T) for binding sites on androgen receptors in mouse kidney cytosol. Their relative binding affinities were T » 6MP > CPA > MPA = P, indicating that MPA, the most biologically potent androgenic progestin, bound to the cytoplasmic androgen receptor with less affinity than 6MP or the antiandrogenic progestin CPA. To further investigate the relationship between binding affinity and biological activity of progestins, an assay using kidney minces was established to study the effect of progestins on the nuclear uptake of the androgen receptor-steroid complex by intact cells. Kidney minces were incubated with [3H]T in the presence or absence of unlabeled steroids. All of the specifically bound nuclear steroid was [3H]T under optimal assay conditions
S
ITUDIES from this and other laboratories indicate that progestins may mimic, inhibit, or potentiate the actions of androgens. These effects have been termed the androgenic, antiandrogenic, and synandrogenic actions of progestins, respectively (1), and have been studied extensively in the reproductive tract and kidney of the mouse. Each of these actions is dependent upon steroid structure and the responsiveness of individual end points (2, 3). Present evidence suggests that all progestins which either mimic or modify androgen action
Received May 10, 1979. Address requests for reprints to: Dr. C. Wayne Bardin, Center for Biomedical Research, The Population Council, The Rockefeller University, New York, New York 10021. * This work was supported in part by NIH Contract NOl-2-2730 and NIH Grant HD-12974. t Present address: Department of Pediatrics, Division of Pediatric Endocrinology, The Johns Hopkins University Hospital, Baltimore, Maryland 21205. $ Recipient of NIH Research Career Development Award HD-00137. 1281 Downloaded from https://academic.oup.com/endo/article-abstract/105/6/1281/2592134 by UB Frankfurt/Main user on 11 May 2018
(at 22 C for 45 min; 5 X 10"" M [yH]T). Nuclear binding was diminished at temperatures above or below 22 C, indicating temperature dependence of nuclear translocation and lability of the androgen receptor. Protease inhibitors, such as leupeptin and antipain, did not significantly stabilize the androgen receptor at 22 C. In contrast to their relative binding affinities in cytosol, the competitive nuclear uptake of progestins (10"'" - 5 X 10 (i M) relative to T in kidney minces was T » MPA = 6 MP > P > CPA. A similar order for the androgenic progestins was obtained with in vivo studies of the relative inhibitory potencies of progestins on nuclear [3H]T uptake by mouse kidney. These observations indicate that the competitive nuclear uptake of progestins relative to T correlates with their androgenic but not with their antiandrogenic and synandrogenic actions. In conclusion, the multiple actions of progestins in mouse kidney cannot be accurately predicted from their binding affinities to androgen receptors. Furthermore, the discrepancy between cytoplasmic binding and nuclear uptake suggested that some property of the receptor in addition to its affinity for a particular steroid is recognized by the cell as a determinant of biological activity. (Endocrinology 105: 1281, 1979)
compete with testosterone (T) for binding sites on the cytoplasmic androgen receptor from kidney (4) and the male reproductive tract (5-7). Although these and other studies (8-10) suggest that some of the biological activities resulting from the interaction of androgens and progestins are mediated by way of the cytoplasmic androgen receptor, to date there is not sufficient evidence to indicate whether all of these effects are modulated via a single receptor. No clear correlation emerged from previous studies (47) designed to correlate the biological activities of androgens and progestins with their relative binding affinities to cytoplasmic receptors of androgen responsive tissues. None of these experiments, however, examined steroid uptake by the nucleus where biological responses are initiated. The emphasis of the present experiments was to determine whether a correlation exists among the androgenic, antiandrogenic, and synandrogenic activities of progestins and their effects on the nuclear uptake of [3H]T.
BROWN, BULLOCK, AND BARDIN
1282
Materials and Methods Steroids [la,2a- 3 H]T (59 Ci/mmol) was obtained from New England Nuclear Corp. (Boston, MA). T and progesterone (P) were purchased from Sigma Chemical Co. (St. Louis, MO). The following steroids were received as gifts: medroxyprogesterone acetate (MPA)1 from the Upjohn Co. (Kalamazoo, MI); cyproterone acetate (CPA) from the Schering Corp. (Berlin, West Germany); 6a-methylprogesterone (6MP) was a gift from Syntex (Palo Alto, CA); and leupeptin (a mixture of iV-acetyl and JV-propyl-L-leucyl-L-leucyl-DL-arginine aldehyde hydrochlorides) and antipain ([(s)-l-carboxy-2-phenylethyl]carbamoylL-arginyl-L-valyl-L-arginine aldehyde) from the United StatesJapan Cooperative Cancer Research Program. Animals Eight- to 12-week-old male C57BL/6J and BALB/cJ mice were from Jackson Laboratories (Bar Harbor, ME). Androgeninsensitive Tfm/Y mice and their normal littermates were from the colony maintained at the Milton S. Hershey Medical Center. Normal mice were castrated via the scrotum under ether anesthesia and were used 18-24 h later for either in vivo or in vitro studies. In vitro nuclear uptake For each sample, six kidneys were minced finely and placed in a 30-ml Corex tube containing 3 ml Eagle's basal medium with Earle's salts (Grand Island Biological, Grand Island, NY). A series of samples was incubated under various concentrations of [3H]T (10~9-10~7 M) with or without unlabeled steroids (10"'°-10"5 M). A 500-fold excess of nonradioactive T was added to assess nonspecific binding. All steroids were added in ethanol (total volume, 20 /xl). Incubations were performed in an atmosphere of 95% 02-5% CO2 in a constant temperature Aquatherm water bath at a shaker speed of 100 rpm. Details on temperature, time of incubation, and steroid concentration in each experiment are provided in the figure legends. The time from the death of the mice to the beginning of the incubation was held constant (10 min) in each experiment. Each study was terminated by immersing the tubes in an ice bath for several minutes before decanting the incubation media. Kidney nuclei were isolated and steroids were extracted, as described below. Nuclear uptake2 was expressed as the specific uptake (total — nonspecific) of radioactivity per mg DNA. In vivo nuclear uptake The in vivo uptake of androgens by kidney nuclei was measured as previously described (11). Briefly, castrated mice were 1
The following abbreviations and trivial names are used: medroxyprogesterone acetate (MPA), 6a-methyl-17a-hydroxy-pregn-4-ene-3,20dione acetate; cyproterone acetate (CPA), 6-chloro-17a-hydroxy-la,2amethylene-pregna-4,6-diene-3,20-dione acetate. 2 In this report, the terms nuclear uptake and relative nuclear uptake are used operationally analogous to the way relative binding affinity is used to compare the binding of tritiated and nonradioactive ligands to a soluble steroid receptor.
Downloaded from https://academic.oup.com/endo/article-abstract/105/6/1281/2592134 by UB Frankfurt/Main user on 11 May 2018
1979 Kndo Vol 105 , Noli
functionally hepatectomized by removal of the intestinal tract just before iv administration of [ J H]T alone or in combination with nonradioactive steroids in 0.25 ml 10% ethanol in saline. The mice were killed 30 min later, since preliminary studies indicated that androgen uptake by renal nuclei was maximal at that time. All animals were killed by cervical dislocation, and the kidneys were rapidly excised. All further tissue processing was done at 0-4 C. Isolation of nuclei Kidneys from three mice were homogenized in 5 ml TMS " buffer (0.03 M Tris-HCl, 3 mM MgCl2, and 0.32 M sucrose, pH 6.9, at 25 C) containing a lOO-'old excess of unlabeled T using a Polytron 10-ST homogenizer (Brinkmann Instruments, Westbury, NY) at a rheostat setting of 5 for 5 sec. The homogenate was filtered through flannel, and the retentate was rinsed with an additional 2 ml TMS buffer. The filtrate was then centri- , fuged at 1000 X g for 10 min in a Sorvall GLC-1 centrifuge (Ivan Sorvall, Inc., Norwalk, CT). The supernatant was aspirated and the pellet was resuspended in 5 ml TMS buffer using 10 strokes of a Dounce homogenizer and recentrifuged at 800 X g for 10 min. Nuclei were resuspended and sedimented three more times: once in TMS buffer containing 0.5% Triton X-100 and twice in TMS buffer. The final pellet was suspended in 3-5 ml TMS buffer. This nuclear fraction was free of cytoplasmic contamination as judged by light microscopy. An aliquot (500 jid) of these nuclei was collected on type HA Millipore filters * (Millipore Corp., Bedford, MA for DNA assay and the remainder was taken for steroid extraction. Extraction and fractionation of radioactive steroids V
Nuclear fractions were extracted with 10 vol methylene chloride. The extracts were washed sequentially with 0.1 N NaOH, 6% acetic acid, and water and dried, and portions were used for measurement of total radioactivity or isolation of individual androgens, as described previously (12). Briefly, T, dihydrotestosterone, and the androstanediols were first separated on silica gel thin layer plates in chloroform-methanol (98:2). Individual steroids were eluted, acetylated, and rechromatographed in benzene-ethyl acetate [80:20; (T acetate) or 90:10 (dihydrotestosterone and androstanediol acetates)]. 14C-Labeled steroids were used to correct for procedural losses. In vitro cytoplasmic binding assay Steroid binding to the cytoplasmic androgen receptor was assayed as described previously (4). Briefly, kidneys were removed and placed in ice-cold TEDG buffer [0.05 M Tris-HCl, 0.1 mM EDTA, and 10% glycerol, pH 7.4, at 25 C (TEG) and 0.25 mM dithiothreitol which was added fresh daily]. All subsequent procedures were carried out at 0-4 C in a cold room. Tissue was weighed, finely minced, and homogenized for 5 sec in 2 vol TEDG using a Polytron 10-ST homogenizer at a rheostat setting of 5. The homogenate was centrifuged at 105,000 x g (av) for 1 h, and the central fraction of the supernatant, diluted 1:1 with TEDG, was used as cytosol (6-8 mg protein/ml). [3H]T (250,000 cpm/10 jttl ethanol) and nonradioactive steroids (in 20 jul ethanol) were added to a series of
Vol. 105, No. 6 Printed in U.S.A.
In Vitro and In Vivo Binding of Progestins to the Androgen Receptor of Mouse Kidney: Correlation with Biological Activities* TERRY R. BROWN,f LESLIE BULLOCKS AND C. WAYNE BARDIN Departments of Medicine and Comparative Medicine, The Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033
ABSTRACT. Progestins possess androgenic, antiandrogenic, and synandrogenic activities on androgen-responsive tissues. Several recent studies suggest that some or all of these actions may be initiated in mouse kidney via progestin interaction with the androgen receptor. All of the progestins studied, including medroxyprogesterone acetate [6a-methyl-17a-hydroxy-pregn-4ene-3,20-dione acetate (MPA)], cyproterone acetate [6-chloro17a-hydroxy-la,2a-methylene-pregna-4,6-diene-3,20-dione acetate (CPA)], 6a-methylprogesterone (6MP), and progesterone (P), effectively competed with [3H]testosterone ([3H]T) for binding sites on androgen receptors in mouse kidney cytosol. Their relative binding affinities were T » 6MP > CPA > MPA = P, indicating that MPA, the most biologically potent androgenic progestin, bound to the cytoplasmic androgen receptor with less affinity than 6MP or the antiandrogenic progestin CPA. To further investigate the relationship between binding affinity and biological activity of progestins, an assay using kidney minces was established to study the effect of progestins on the nuclear uptake of the androgen receptor-steroid complex by intact cells. Kidney minces were incubated with [3H]T in the presence or absence of unlabeled steroids. All of the specifically bound nuclear steroid was [3H]T under optimal assay conditions
S
ITUDIES from this and other laboratories indicate that progestins may mimic, inhibit, or potentiate the actions of androgens. These effects have been termed the androgenic, antiandrogenic, and synandrogenic actions of progestins, respectively (1), and have been studied extensively in the reproductive tract and kidney of the mouse. Each of these actions is dependent upon steroid structure and the responsiveness of individual end points (2, 3). Present evidence suggests that all progestins which either mimic or modify androgen action
Received May 10, 1979. Address requests for reprints to: Dr. C. Wayne Bardin, Center for Biomedical Research, The Population Council, The Rockefeller University, New York, New York 10021. * This work was supported in part by NIH Contract NOl-2-2730 and NIH Grant HD-12974. t Present address: Department of Pediatrics, Division of Pediatric Endocrinology, The Johns Hopkins University Hospital, Baltimore, Maryland 21205. $ Recipient of NIH Research Career Development Award HD-00137. 1281 Downloaded from https://academic.oup.com/endo/article-abstract/105/6/1281/2592134 by UB Frankfurt/Main user on 11 May 2018
(at 22 C for 45 min; 5 X 10"" M [yH]T). Nuclear binding was diminished at temperatures above or below 22 C, indicating temperature dependence of nuclear translocation and lability of the androgen receptor. Protease inhibitors, such as leupeptin and antipain, did not significantly stabilize the androgen receptor at 22 C. In contrast to their relative binding affinities in cytosol, the competitive nuclear uptake of progestins (10"'" - 5 X 10 (i M) relative to T in kidney minces was T » MPA = 6 MP > P > CPA. A similar order for the androgenic progestins was obtained with in vivo studies of the relative inhibitory potencies of progestins on nuclear [3H]T uptake by mouse kidney. These observations indicate that the competitive nuclear uptake of progestins relative to T correlates with their androgenic but not with their antiandrogenic and synandrogenic actions. In conclusion, the multiple actions of progestins in mouse kidney cannot be accurately predicted from their binding affinities to androgen receptors. Furthermore, the discrepancy between cytoplasmic binding and nuclear uptake suggested that some property of the receptor in addition to its affinity for a particular steroid is recognized by the cell as a determinant of biological activity. (Endocrinology 105: 1281, 1979)
compete with testosterone (T) for binding sites on the cytoplasmic androgen receptor from kidney (4) and the male reproductive tract (5-7). Although these and other studies (8-10) suggest that some of the biological activities resulting from the interaction of androgens and progestins are mediated by way of the cytoplasmic androgen receptor, to date there is not sufficient evidence to indicate whether all of these effects are modulated via a single receptor. No clear correlation emerged from previous studies (47) designed to correlate the biological activities of androgens and progestins with their relative binding affinities to cytoplasmic receptors of androgen responsive tissues. None of these experiments, however, examined steroid uptake by the nucleus where biological responses are initiated. The emphasis of the present experiments was to determine whether a correlation exists among the androgenic, antiandrogenic, and synandrogenic activities of progestins and their effects on the nuclear uptake of [3H]T.
BROWN, BULLOCK, AND BARDIN
1282
Materials and Methods Steroids [la,2a- 3 H]T (59 Ci/mmol) was obtained from New England Nuclear Corp. (Boston, MA). T and progesterone (P) were purchased from Sigma Chemical Co. (St. Louis, MO). The following steroids were received as gifts: medroxyprogesterone acetate (MPA)1 from the Upjohn Co. (Kalamazoo, MI); cyproterone acetate (CPA) from the Schering Corp. (Berlin, West Germany); 6a-methylprogesterone (6MP) was a gift from Syntex (Palo Alto, CA); and leupeptin (a mixture of iV-acetyl and JV-propyl-L-leucyl-L-leucyl-DL-arginine aldehyde hydrochlorides) and antipain ([(s)-l-carboxy-2-phenylethyl]carbamoylL-arginyl-L-valyl-L-arginine aldehyde) from the United StatesJapan Cooperative Cancer Research Program. Animals Eight- to 12-week-old male C57BL/6J and BALB/cJ mice were from Jackson Laboratories (Bar Harbor, ME). Androgeninsensitive Tfm/Y mice and their normal littermates were from the colony maintained at the Milton S. Hershey Medical Center. Normal mice were castrated via the scrotum under ether anesthesia and were used 18-24 h later for either in vivo or in vitro studies. In vitro nuclear uptake For each sample, six kidneys were minced finely and placed in a 30-ml Corex tube containing 3 ml Eagle's basal medium with Earle's salts (Grand Island Biological, Grand Island, NY). A series of samples was incubated under various concentrations of [3H]T (10~9-10~7 M) with or without unlabeled steroids (10"'°-10"5 M). A 500-fold excess of nonradioactive T was added to assess nonspecific binding. All steroids were added in ethanol (total volume, 20 /xl). Incubations were performed in an atmosphere of 95% 02-5% CO2 in a constant temperature Aquatherm water bath at a shaker speed of 100 rpm. Details on temperature, time of incubation, and steroid concentration in each experiment are provided in the figure legends. The time from the death of the mice to the beginning of the incubation was held constant (10 min) in each experiment. Each study was terminated by immersing the tubes in an ice bath for several minutes before decanting the incubation media. Kidney nuclei were isolated and steroids were extracted, as described below. Nuclear uptake2 was expressed as the specific uptake (total — nonspecific) of radioactivity per mg DNA. In vivo nuclear uptake The in vivo uptake of androgens by kidney nuclei was measured as previously described (11). Briefly, castrated mice were 1
The following abbreviations and trivial names are used: medroxyprogesterone acetate (MPA), 6a-methyl-17a-hydroxy-pregn-4-ene-3,20dione acetate; cyproterone acetate (CPA), 6-chloro-17a-hydroxy-la,2amethylene-pregna-4,6-diene-3,20-dione acetate. 2 In this report, the terms nuclear uptake and relative nuclear uptake are used operationally analogous to the way relative binding affinity is used to compare the binding of tritiated and nonradioactive ligands to a soluble steroid receptor.
Downloaded from https://academic.oup.com/endo/article-abstract/105/6/1281/2592134 by UB Frankfurt/Main user on 11 May 2018
1979 Kndo Vol 105 , Noli
functionally hepatectomized by removal of the intestinal tract just before iv administration of [ J H]T alone or in combination with nonradioactive steroids in 0.25 ml 10% ethanol in saline. The mice were killed 30 min later, since preliminary studies indicated that androgen uptake by renal nuclei was maximal at that time. All animals were killed by cervical dislocation, and the kidneys were rapidly excised. All further tissue processing was done at 0-4 C. Isolation of nuclei Kidneys from three mice were homogenized in 5 ml TMS " buffer (0.03 M Tris-HCl, 3 mM MgCl2, and 0.32 M sucrose, pH 6.9, at 25 C) containing a lOO-'old excess of unlabeled T using a Polytron 10-ST homogenizer (Brinkmann Instruments, Westbury, NY) at a rheostat setting of 5 for 5 sec. The homogenate was filtered through flannel, and the retentate was rinsed with an additional 2 ml TMS buffer. The filtrate was then centri- , fuged at 1000 X g for 10 min in a Sorvall GLC-1 centrifuge (Ivan Sorvall, Inc., Norwalk, CT). The supernatant was aspirated and the pellet was resuspended in 5 ml TMS buffer using 10 strokes of a Dounce homogenizer and recentrifuged at 800 X g for 10 min. Nuclei were resuspended and sedimented three more times: once in TMS buffer containing 0.5% Triton X-100 and twice in TMS buffer. The final pellet was suspended in 3-5 ml TMS buffer. This nuclear fraction was free of cytoplasmic contamination as judged by light microscopy. An aliquot (500 jid) of these nuclei was collected on type HA Millipore filters * (Millipore Corp., Bedford, MA for DNA assay and the remainder was taken for steroid extraction. Extraction and fractionation of radioactive steroids V
Nuclear fractions were extracted with 10 vol methylene chloride. The extracts were washed sequentially with 0.1 N NaOH, 6% acetic acid, and water and dried, and portions were used for measurement of total radioactivity or isolation of individual androgens, as described previously (12). Briefly, T, dihydrotestosterone, and the androstanediols were first separated on silica gel thin layer plates in chloroform-methanol (98:2). Individual steroids were eluted, acetylated, and rechromatographed in benzene-ethyl acetate [80:20; (T acetate) or 90:10 (dihydrotestosterone and androstanediol acetates)]. 14C-Labeled steroids were used to correct for procedural losses. In vitro cytoplasmic binding assay Steroid binding to the cytoplasmic androgen receptor was assayed as described previously (4). Briefly, kidneys were removed and placed in ice-cold TEDG buffer [0.05 M Tris-HCl, 0.1 mM EDTA, and 10% glycerol, pH 7.4, at 25 C (TEG) and 0.25 mM dithiothreitol which was added fresh daily]. All subsequent procedures were carried out at 0-4 C in a cold room. Tissue was weighed, finely minced, and homogenized for 5 sec in 2 vol TEDG using a Polytron 10-ST homogenizer at a rheostat setting of 5. The homogenate was centrifuged at 105,000 x g (av) for 1 h, and the central fraction of the supernatant, diluted 1:1 with TEDG, was used as cytosol (6-8 mg protein/ml). [3H]T (250,000 cpm/10 jttl ethanol) and nonradioactive steroids (in 20 jul ethanol) were added to a series of
