0021 -972X/79/4804-0655$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1979 by The Endocrine Society
Vol. 48, No. 4 Printed in U.S.A.
Parathyroid Hormone Inhibitors: Comparison of Biological Activity in Bone- and Skin-Derived Tissue* STEVEN R. GOLDRING, JANE E. MAHAFFEY,f MICHAEL ROSENBLATT^ JEANMICHEL DAYER,§ JOHN T. POTTS, JR., AND STEPHEN M. KRANE Department of Medicine, Harvard Medical School, and the Medical Services (Arthritis and Endocrine Units), Massachusetts General Hospital, Boston, 02114; and the New England Deaconess Hospital, Boston, Massachusetts 02215
ABSTRACT. To evaluate the biological properties of parathyroid hormone (PTH) and two synthetic inhibitors of PTH, monolayer cell cultures derived from two human tissues were utilized. One cell culture system was prepared from human giant cell tumors of bone and the other from human neonatal foreskin. The level of cAMP was increased in cells obtained from both of these tissues when the cell cultures were incubated with PTH. The apparent affinity constants for PTH (1.2 x 10~8 and 1.6 x 10~8 M in dermal and tumor-derived cells, respectively) and the concentrations of the synthetic PTH analogs which produced 50% inhibition of PTH-induced increases in cAMP content in the two human cell cultures (5 x 10~8 and 8 X 10~8 M for the
analog [Nle-8, Nle-18, Tyr-34]bovine PTH-(3-34) amide in dermal and tumor-derived cells, respectively) were nearly identical to the values previously observed in studies employing membranes prepared from canine renal tissue. Although not regarded as a PTH target tissue, fibroblasts from human neonatal foreskin possessed PTH receptors comparable to those of bone and kidney tissue. This study extends the evaluation of parathyroid inhibitors to cells in monolayer culture and illustrates that such cell culture systems provide a model for investigation of PTH receptor interactions in human tissues. (J Clin Endocrinol Metab 48: 655, 1979)
S
TRUCTURE-activity studies with parathyroid hormone (PTH) and synthetic fragments of the hormone using a PTH-responsive adenylate cyclase system prepared from renal cortical membranes (1) have identified a region of the PTH molecule which does not stimulate adenylate cyclase, yet inhibits the PTH-stim ulated increase in adenylate cyclase activity. Subsequent studies have demonstrated a 30-fold increase in inhibitory potency when activity-enhancing modifications were imposed on the inhibitory core (2). Coincubation of the renal membranes with one of the synthetic inhibitory analogs, [Nle-8, Nle-18, Tyr-34]bovine PTH-(3-34) amide, and native PTH at equimolar concentrations caused approximately 50% inhibition of PTH-stimulated adenylate cyclase activity. This hormone antagonist lacked PTH-like agonist activity at doses 500-fold those
Received September 18, 1978. Address requests for reprints to: Dr. Steven R. Goldring, The Arthritis Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. * This work was supported in part by NIAMDD Grants AM-03564, AM-04501, and AM-11794. f Teaching and Research Scholar of the American College of Physicians. % Recipient of a National Research Service Award and a Research Fellowship from the Medical Foundation funded by the Godfrey M. Hyams Trust. § Supported by Fond National Suisse de Recherche Scientifique.
required to demonstrate maximal adenylate cyclase activation with native PTH and, at appropriate concentrations, it completely suppressed PTH activation of membrane adenylate cyclase. The kinetics of this inhibition were characteristic of a competitive inhibitor. To evaluate the biological properties of this inhibitor in another known major PTH target tissue, bone, we have undertaken in vitro studies similar to those performed with kidney membranes using monolayer cultures of cells derived from human giant cell tumors of bone. We have previously shown that these cells increase cAMP content when incubated with PTH (3,4). A similar pattern of response has been demonstrated in cells derived from murine skeletal tissue (5, 6). In contrast to the studies with fragmented renal plasma membranes, the studies with monolayer cell cultures assess the biological effects of PTH and PTH inhibitors on intact living cells. Cells derived from human neonatal foreskin were also evaluated after preliminary studies indicated that these cells increased cAMP content when exposed to PTH (4). Because of their rapid doubling time in culture and the reproducibility of the pattern of hormone response, these cells have advantages for extended use in investigation of the mechanisms of hormonal action. Analysis of the effects of PTH and the PTH inhibitors on the cAMP response in these monolayer cell cultures provides an
655
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GOLDRING ET AL.
656
opportunity for comparison of structural requirements for binding to receptors and activation of adenylate cyclase in renal, skeletal, and dermal tissue. Materials and Methods Tissue preparation and culture procedures Sterile specimens of tumor tissue were obtained at the time of surgery from patients undergoing therapeutic giant cell tumor resection and allograft transplantation (7) and were kindly provided by Dr. Henry J. Mankin. The diagnosis of giant cell tumor of bone was established on the basis of clinical history, tumor site, and histological evaluation. A single sample of foreskin tissue was obtained at the time of circumcision of a normal male infant and served as the source of cells for all of the subsequent studies. Tissues were prepared for culture by dispersion with trypsin-EDTA and clostridial collagenase, and cells were cultured in plastic petri dishes, as previously described (8). After primary culture and growth to confluence in 10-cm diameter plastic petri dishes, the cells were subcultured, as previously described (3, 4). Before incubation with hormone, cells were transferred to plastic trays containing 24 individual wells, each 1.6 cm in diameter (Costar), at a density of 5 X 105 cells/well. Hormone incubation Medium was first removed from the cells and replaced with buffer solution (3, 4) containing native hormone or its analogs. The trays were then partially immersed in a water bath at 37 C and incubated for 10 min, after which trichloroacetic acid (TCA) at 2 C was added to a final concentration of 8%. The trays were then frozen in liquid nitrogen and stored at —30 C. Frozen samples (cells plus medium) in 8% TCA were thawed at room temperature, the cells were scraped with a plastic policeman, and the contents were transferred to polystyrene tubes and centrifuged at 4 C at 600 X g for 20 min. The supernatant solutions were then removed for cAMP assay. Pellets were stored at —30 C and assayed for DNA content using a fluorometric micromethod (9). Hormone preparations Solutions of the native hormone or its analogs were prepared by dissolving lyophilized samples of either highly purified bovine PTH-(l-84) [bPTH-(l-84)J or the inhibitory analogs (1, 2) in buffer medium, and the concentrations were adjusted by serial dilution. Native bPTH (3000 Medical Research Council U/mg), a gift from Dr. Henry Keutmann, was prepared from the TCA precipitate of bovine parathyroid glands and purified by gel filtration, followed by ion exchange chromatography on carboxymethyl-cellulose (10). Synthetic inhibitory analogs of PTH were prepared by a modification (11) of the Merrifield method of solid phase peptide synthesis (12). Details of their purification and chemical analysis have been reported previously (11, 13, 14). Media containing prostaglandin E2 (PGE2) were prepared by diluting in the buffer solution a stock solution (1 mg/ml) of PGE2 (Upjohn) in ethanol.
JCE&M V0U8
1979 No 4
Assays The supernatant solutions (1 ml) obtained after centrifugation of TCA-treated samples were added to columns containing 2 ml Dowex 50W-X4 resin (200-400 mesh; H + form) equilibrated with 0.1 N HC1, and cAMP was eluted as previously described (4). The samples were lyophilized for storage and then dissolved in 50 mM sodium acetate, pH 6.2 and cAMP content was determined by RIA (Schwarz-Mann). Corrections for recovery were made by the addition of a [3H]cAMP standard to the original samples. Recovery of cAMP ranged from 60-90%. Results Experiments with giant cell tumors Cells from three giant cell tumors studied in the first passage were exposed to several concentrations of native bPTH-(l-84). The apparent affinity constants (Kms) for native PTH in these cells (concentration required for 50% maximal increase in cAMP content) were: 1.4 X 10~8, 1.6 X 10~8, and 1.8 X 10~8 M. Representative data from cells cultured from one tumor are shown in Fig 1. Cells from this tumor did not increase cAMP content when incubated with the inhibitor bPTH-(3-34) or the inhibitor [Nle-8, Nle-18, Tyr-34]bPTH-(3-34) amide at concentrations as high as 5 x 10~5 M (a concentration at least 20-fold that required to cause a maximal increase in
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bPTH (M) FIG. 1. Response of cultured giant cell tumor cells to bPTH-(l-84). The levels of accumulated cAMP were measured after incubation of the cells for 10 min in the absence and presence of different concentrations of bPTH-(l-84). The apparent Km, indicated by the arrow, is 1.4 x 10~8 M. In this and subsequent figures, brackets indicate SEMS for triplicate wells.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 03:02 For personal use only. No other uses without permission. . All rights reserved.
SYNTHETIC PTH INHIBITORS
cAMP content with native bPTH). However, both inhibitors did prevent the increase in cAMP content normally observed in cells exposed to bPTH-(l-84) (1 X 10~7 M). As shown in Fig. 2, concentrations required to cause 50% inhibition of PTH-stimulated increase in cAMP content were 3 X 1(T7 M for bPTH-(3-34) and 5 X 10"8 M for [Nle8, Nle-18, Tyr-34]bPTH-(3-34) amide.
657
I4r 12 10 to
Experiments with fibroblasts Cells cultured from neonatal foreskin were exposed to several concentrations of native bPTH-(l-84). Figure 3 illustrates the dose-dependent relationship for concentrations of bPTH-(l-84) and cAMP content. The apparent Km for native bPTH was 1.2 X 10~8 M in cells in early passage. These cells did not show increases in cAMP content when incubated with the inhibitor [Nle-8, Nle18, Tyr-34]bPTH-(3-34) amide. However, accumulation of cAMP in cells exposed to native bPTH (1 X 10~7 M) was inhibited in the presence of [Nle-8, Nle-18, Tyr34]bPTH-(3-34) amide, as shown in Fig. 4. The concentration of this inhibitor which produced a 50% inhibition of PTH-stimulated increase in cAMP content was 8 X 10"8 M. Specificity of response to PTH The specificity of the antagonism produced by the synthetic analogs of PTH was assessed by incubation of cells from the giant cell tumors and cells from foreskin
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Vol. 48, No. 4 Printed in U.S.A.
Parathyroid Hormone Inhibitors: Comparison of Biological Activity in Bone- and Skin-Derived Tissue* STEVEN R. GOLDRING, JANE E. MAHAFFEY,f MICHAEL ROSENBLATT^ JEANMICHEL DAYER,§ JOHN T. POTTS, JR., AND STEPHEN M. KRANE Department of Medicine, Harvard Medical School, and the Medical Services (Arthritis and Endocrine Units), Massachusetts General Hospital, Boston, 02114; and the New England Deaconess Hospital, Boston, Massachusetts 02215
ABSTRACT. To evaluate the biological properties of parathyroid hormone (PTH) and two synthetic inhibitors of PTH, monolayer cell cultures derived from two human tissues were utilized. One cell culture system was prepared from human giant cell tumors of bone and the other from human neonatal foreskin. The level of cAMP was increased in cells obtained from both of these tissues when the cell cultures were incubated with PTH. The apparent affinity constants for PTH (1.2 x 10~8 and 1.6 x 10~8 M in dermal and tumor-derived cells, respectively) and the concentrations of the synthetic PTH analogs which produced 50% inhibition of PTH-induced increases in cAMP content in the two human cell cultures (5 x 10~8 and 8 X 10~8 M for the
analog [Nle-8, Nle-18, Tyr-34]bovine PTH-(3-34) amide in dermal and tumor-derived cells, respectively) were nearly identical to the values previously observed in studies employing membranes prepared from canine renal tissue. Although not regarded as a PTH target tissue, fibroblasts from human neonatal foreskin possessed PTH receptors comparable to those of bone and kidney tissue. This study extends the evaluation of parathyroid inhibitors to cells in monolayer culture and illustrates that such cell culture systems provide a model for investigation of PTH receptor interactions in human tissues. (J Clin Endocrinol Metab 48: 655, 1979)
S
TRUCTURE-activity studies with parathyroid hormone (PTH) and synthetic fragments of the hormone using a PTH-responsive adenylate cyclase system prepared from renal cortical membranes (1) have identified a region of the PTH molecule which does not stimulate adenylate cyclase, yet inhibits the PTH-stim ulated increase in adenylate cyclase activity. Subsequent studies have demonstrated a 30-fold increase in inhibitory potency when activity-enhancing modifications were imposed on the inhibitory core (2). Coincubation of the renal membranes with one of the synthetic inhibitory analogs, [Nle-8, Nle-18, Tyr-34]bovine PTH-(3-34) amide, and native PTH at equimolar concentrations caused approximately 50% inhibition of PTH-stimulated adenylate cyclase activity. This hormone antagonist lacked PTH-like agonist activity at doses 500-fold those
Received September 18, 1978. Address requests for reprints to: Dr. Steven R. Goldring, The Arthritis Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. * This work was supported in part by NIAMDD Grants AM-03564, AM-04501, and AM-11794. f Teaching and Research Scholar of the American College of Physicians. % Recipient of a National Research Service Award and a Research Fellowship from the Medical Foundation funded by the Godfrey M. Hyams Trust. § Supported by Fond National Suisse de Recherche Scientifique.
required to demonstrate maximal adenylate cyclase activation with native PTH and, at appropriate concentrations, it completely suppressed PTH activation of membrane adenylate cyclase. The kinetics of this inhibition were characteristic of a competitive inhibitor. To evaluate the biological properties of this inhibitor in another known major PTH target tissue, bone, we have undertaken in vitro studies similar to those performed with kidney membranes using monolayer cultures of cells derived from human giant cell tumors of bone. We have previously shown that these cells increase cAMP content when incubated with PTH (3,4). A similar pattern of response has been demonstrated in cells derived from murine skeletal tissue (5, 6). In contrast to the studies with fragmented renal plasma membranes, the studies with monolayer cell cultures assess the biological effects of PTH and PTH inhibitors on intact living cells. Cells derived from human neonatal foreskin were also evaluated after preliminary studies indicated that these cells increased cAMP content when exposed to PTH (4). Because of their rapid doubling time in culture and the reproducibility of the pattern of hormone response, these cells have advantages for extended use in investigation of the mechanisms of hormonal action. Analysis of the effects of PTH and the PTH inhibitors on the cAMP response in these monolayer cell cultures provides an
655
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 03:02 For personal use only. No other uses without permission. . All rights reserved.
GOLDRING ET AL.
656
opportunity for comparison of structural requirements for binding to receptors and activation of adenylate cyclase in renal, skeletal, and dermal tissue. Materials and Methods Tissue preparation and culture procedures Sterile specimens of tumor tissue were obtained at the time of surgery from patients undergoing therapeutic giant cell tumor resection and allograft transplantation (7) and were kindly provided by Dr. Henry J. Mankin. The diagnosis of giant cell tumor of bone was established on the basis of clinical history, tumor site, and histological evaluation. A single sample of foreskin tissue was obtained at the time of circumcision of a normal male infant and served as the source of cells for all of the subsequent studies. Tissues were prepared for culture by dispersion with trypsin-EDTA and clostridial collagenase, and cells were cultured in plastic petri dishes, as previously described (8). After primary culture and growth to confluence in 10-cm diameter plastic petri dishes, the cells were subcultured, as previously described (3, 4). Before incubation with hormone, cells were transferred to plastic trays containing 24 individual wells, each 1.6 cm in diameter (Costar), at a density of 5 X 105 cells/well. Hormone incubation Medium was first removed from the cells and replaced with buffer solution (3, 4) containing native hormone or its analogs. The trays were then partially immersed in a water bath at 37 C and incubated for 10 min, after which trichloroacetic acid (TCA) at 2 C was added to a final concentration of 8%. The trays were then frozen in liquid nitrogen and stored at —30 C. Frozen samples (cells plus medium) in 8% TCA were thawed at room temperature, the cells were scraped with a plastic policeman, and the contents were transferred to polystyrene tubes and centrifuged at 4 C at 600 X g for 20 min. The supernatant solutions were then removed for cAMP assay. Pellets were stored at —30 C and assayed for DNA content using a fluorometric micromethod (9). Hormone preparations Solutions of the native hormone or its analogs were prepared by dissolving lyophilized samples of either highly purified bovine PTH-(l-84) [bPTH-(l-84)J or the inhibitory analogs (1, 2) in buffer medium, and the concentrations were adjusted by serial dilution. Native bPTH (3000 Medical Research Council U/mg), a gift from Dr. Henry Keutmann, was prepared from the TCA precipitate of bovine parathyroid glands and purified by gel filtration, followed by ion exchange chromatography on carboxymethyl-cellulose (10). Synthetic inhibitory analogs of PTH were prepared by a modification (11) of the Merrifield method of solid phase peptide synthesis (12). Details of their purification and chemical analysis have been reported previously (11, 13, 14). Media containing prostaglandin E2 (PGE2) were prepared by diluting in the buffer solution a stock solution (1 mg/ml) of PGE2 (Upjohn) in ethanol.
JCE&M V0U8
1979 No 4
Assays The supernatant solutions (1 ml) obtained after centrifugation of TCA-treated samples were added to columns containing 2 ml Dowex 50W-X4 resin (200-400 mesh; H + form) equilibrated with 0.1 N HC1, and cAMP was eluted as previously described (4). The samples were lyophilized for storage and then dissolved in 50 mM sodium acetate, pH 6.2 and cAMP content was determined by RIA (Schwarz-Mann). Corrections for recovery were made by the addition of a [3H]cAMP standard to the original samples. Recovery of cAMP ranged from 60-90%. Results Experiments with giant cell tumors Cells from three giant cell tumors studied in the first passage were exposed to several concentrations of native bPTH-(l-84). The apparent affinity constants (Kms) for native PTH in these cells (concentration required for 50% maximal increase in cAMP content) were: 1.4 X 10~8, 1.6 X 10~8, and 1.8 X 10~8 M. Representative data from cells cultured from one tumor are shown in Fig 1. Cells from this tumor did not increase cAMP content when incubated with the inhibitor bPTH-(3-34) or the inhibitor [Nle-8, Nle-18, Tyr-34]bPTH-(3-34) amide at concentrations as high as 5 x 10~5 M (a concentration at least 20-fold that required to cause a maximal increase in
Q
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Q)
-
2
< o
o Li —il-
vIO
10,-9
IO~8
10,-7
10"
bPTH (M) FIG. 1. Response of cultured giant cell tumor cells to bPTH-(l-84). The levels of accumulated cAMP were measured after incubation of the cells for 10 min in the absence and presence of different concentrations of bPTH-(l-84). The apparent Km, indicated by the arrow, is 1.4 x 10~8 M. In this and subsequent figures, brackets indicate SEMS for triplicate wells.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 03:02 For personal use only. No other uses without permission. . All rights reserved.
SYNTHETIC PTH INHIBITORS
cAMP content with native bPTH). However, both inhibitors did prevent the increase in cAMP content normally observed in cells exposed to bPTH-(l-84) (1 X 10~7 M). As shown in Fig. 2, concentrations required to cause 50% inhibition of PTH-stimulated increase in cAMP content were 3 X 1(T7 M for bPTH-(3-34) and 5 X 10"8 M for [Nle8, Nle-18, Tyr-34]bPTH-(3-34) amide.
657
I4r 12 10 to
Experiments with fibroblasts Cells cultured from neonatal foreskin were exposed to several concentrations of native bPTH-(l-84). Figure 3 illustrates the dose-dependent relationship for concentrations of bPTH-(l-84) and cAMP content. The apparent Km for native bPTH was 1.2 X 10~8 M in cells in early passage. These cells did not show increases in cAMP content when incubated with the inhibitor [Nle-8, Nle18, Tyr-34]bPTH-(3-34) amide. However, accumulation of cAMP in cells exposed to native bPTH (1 X 10~7 M) was inhibited in the presence of [Nle-8, Nle-18, Tyr34]bPTH-(3-34) amide, as shown in Fig. 4. The concentration of this inhibitor which produced a 50% inhibition of PTH-stimulated increase in cAMP content was 8 X 10"8 M. Specificity of response to PTH The specificity of the antagonism produced by the synthetic analogs of PTH was assessed by incubation of cells from the giant cell tumors and cells from foreskin
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