JOURNAL OF BONE AND MINERAL RESE4RCH Volume 5, Number 11, 1990 Mary Ann Liebert, Inc., Publishers
Effects of Androgens on Parathyroid Hormone and Interleukin-1-Stimulated Prostaglandin Production in Cultured Neonatal Mouse Calvariae CAROL C. PILBEAM and LAWRENCE G . RAISZ
ABSTRACT In this study we show direct inhibitory effects on prostaglandin E2 (PGE,) production by the androgens, testosterone (T) and dihydrotestosterone (DHT), in cultured neonatal mouse calvariae. After 24 h of preculture with or without androgens, bones were treated with bovine (&34)-parathyroid hormone (PTH) or recombinant human interleukin-la (IL-1). During preculture androgens decreased PGE, release only in those experiments in which control PGE, was high. PTH increased medium PGE, 9-fold at 24 h, and lo-" M T inhibited this increase by 50%. Treatment with IL-1 for 24 h increased medium PGE, 19- to 22-fold, and lo-'' M T and DHT inhibited this increase by 60 and 70'70, respectively. T did not significantly affect the PTH-stimulated release of previously incorporated 4sCaor alter the PTH inhibition of incorporation of [JH]prolineinto collagenase-digestible protein. IL-1 stimulated 45Carelease by 60-80%, and small but significant reductions of 20-30070 were seen with T and DHT. This study shows that T and DHT have direct effects on bone at physiologic concentrations, similar to our previous study in which PTH-stimulated PGE, production in the same culture system was inhibited by physiologic concentrations of 17@-estradiol,and suggests that prostaglandins may mediate some of the effects of androgens in vivo.
INTRODUCTION are potent regulators of bone metabolism in vivo. Deficiency of either is associated with reduced bone mass and increased risk for osteoporosis in both humans and Men with hypogonadism secondary to an isolated deficiency of hypothalamic gonadotropin releasing hormone have decreased cortical and spinal trabecular bone density compared to normal individualsI6) and have shown increases in bone density during gonadal steroid treatment.I7) As in women after estrogen w i t h d r a ~ a l , ' ~androgen .~) deficiency may be associated with increased bone resorption and loss of bone mass compared to normal controls, ID) and the increased turnover may be reduced by androgen replacement . ( I 1 ) However, other studies have suggested that the primary abnormality associated with hypogonadism in men may be
A
NDROGENS AND ESTROGENS
impaired bone formation.(b' " 1 3 ) Androgens also influence bone metabolism in women as well as in men,(I41 5 ) and anabolic steroids have been used to treat osteoporotic women.('b 1 7 ) The mechanism by which androgens and estrogens influence bone metabolism in vivo is unknown. Estrogen has been shown to inhibit parathyroid hormone (PTH)-stimulated bone resorption in postmenopausal women with primary hyperparathyroidism.(18-'0)An in vivo study suggested increased sensitivity of bone to PTH in castrated male mice. [ ' I ) Similar to other androgen target tissues, human bone is capable of metabolizing testosterone (T) to dihydrotestosterone (DHT).IZ2)Direct effects of sex hormones on bone metabolism have been suggested by recent studies that have demonstrated both estrogen and androgen receptors in cultured human osteoblastlike cells from both males and females(z3 and in human osteosarcoma cells."' z6)
Division of Endocrinology and Metabolism, Department of Medicine, University of Connecticut Health Center, Farmington, C T 06032.
1183
PILBEAM AND RAISZ
1184
Both estrogen and androgen have been shown to affect cell proliferation and alkaline phosphatase production in osteoblastlike cell^.'^'-^'' Both have also been shown to inand to decrease PTHcrease type I collagen mRNA(25~'6~30) stimulated CAMP p r o d ~ c t i o n ( ~ *in~ ~human ) osteosarcoma cells. However, attempts to show a direct effect of androgen at physiologic concentrations on bone collagen synthesisi33'and on PTH-stimulated bone in organ culture have been unsuccessful. Several studies have suggested that the actions of androgens and estrogens may be mediated by autocrine-paracrine factors. Studies in osteoblastlike cells have shown that estrogen can increase secretion of insulinlike growth and mRNA for insulinlike growth factor-I (IGFI)"') and that both androgen and estrogen can increase In mRNA for transforming growth factor /3 (TGF-fi).i25.26) vivo studies have related sex steroid deficiency to increased interleukin-l (IL-1) production by monocytes in both men and ~ o m e n . ( ~ ~We - ' ~have ) shown that oophorectomy increased prostaglandin E2 (PGE,) production by excised rat ~alvariae'~') and that estrogen treatment in vitro reduced PTH-stimulated PGE, production by neonatal mouse cal~ a r i a e . ' ~ In ' ) the present study we demonstrate a direct effect of physiologic concentrations of T and DHT on PTHand IL-I-stimulated PGE, production and on IL-I-stimulated resorption in cultured neonatal mouse calvariae.
MATERIALS AND METHODS Timed pregnant CDI mice (Charles River, Wilmington, MA) were injected on day 16 of gestation with 50 pCi of Y a (New England Nuclear, Boston, MA) to label fetal bones in utero. Calvariae were excised from the neonates 7 days after birth, and pieces of parietal bone about 2 x 3 mm were dissected free of all sutures. Bones were incubated in 12-well plates (Costar, Cambridge, MA) in 1 ml medium on a rocker platform at 37°C in a humidified atmosphere of 5% CO, in air. Bones from each litter were
TABLE1. EFFECTS OF TESTOSTERONE (T) OR DIHYDROTESTOSTERONE (DHT) ON PROSTAGLANDIN E, (PGE,) RELEASE^
distributed equally throughout the different treatment groups. All experiments had stimulated and unstimulated control groups, but not all androgen doses were in every experiment. The basic culture medium was phenol red-free BGJb (GIBCO, Grand Island, NY) with 1 mg/ml of bovine serum albumin (BSA, radioimmunoassay grade, Sigma, St. Louis, MO), 1 mM proline (Sigma), 0.4 mM 1glutamine (Sigma), 0.6 mM I-ascorbic acid (Sigma), and 3 mM phosphate. Bones were precultured for 24 h with or without T (Sigma) or DHT (Sigma) and then transferred to similar media with or without 2.5 nM synthetic bovine (1-34)-PTH (Bachem, Torrance, CA) or 0.1 ng/ml of recombinant human 1L-la (kindly provided by Dr. P. Lomedico, Hoffmann-LaRoche, Nutley, NJ) and cultured for another 48 h with a medium change after 24 h. Androgens were dissolved in ethanol, and media for all treatment and control groups contained 0.1070 ethanol. Medium was assayed at the end of preculture and at 24 and 48 h after the addition of PTH or IL-I for PGE, using radioimmunoassay (RIA, lower limit of detection 0.10 nM), as described previously. ( 4 1 ) PGE, antibodies were kindly provided by Dr. N.H.L. Hunt, Canberra, Australia. To prevent the interference of W a with 3H in the RIA, medium calcium was precipitated before RIA with ammonium oxalate (1 pl saturated solution added to 20 p1 medium, incubated 1 h at 37"C, and centrifuged at 1500 cpm and 4°C for 15 minutes). The W a content of the medium at 24 and 48 h after addition of PTH or IL-1 and of the 5 % (v/v) trichloroacetic acid (TCA) extracts of the bones was measured by liquid scintillation counting and used to calculate the total W a and the cumulative percentage of "'Ca released, as described previously.(42) To assess collagen and noncollagen protein synthesis, bones were pulsed with 5 pCi/ml of [3H]proline (New England Nuclear) for the last 2 h of a 48 h culture. Bones were extracted with 5% TCA, acetone, and ether, dried, weighed, homogenized, and treated with purified bacterial collagenase (Worthington Biochemical Corp., Freehold, NJ) to measure incorporation of [3H]proline in collage-
OF TESTOSTERONE (T) ON MEDIUM TABLE2. EFFECTS PROSTAGLANDIN E, (PGE,) CONCENTRATION^
Treatment
N
Medium PGE, concentration fnW
Control
35 42 11 24 24 19 18 14
0.5 f 0.lb 4.6 f 0.5 3.3 f 1.0 2.6 f 0.5 2.2 f 0.3b 2.8 f 0.4 2.5 f 0.3 3.9 f 0.7
Medium PGE, concentration, nM (N)
> 6.0nM
Treatment
Control
Control T lo-" M T lo-'' M
7.3 4.1 4.9
Control DHT lo-" M DHT lo-" M
7.4 f 0.4 (44) 3.6 f 0.2 (24)b 3.9 f 0.3 (24)b
Control
0.4 (55) f 0.3 (20)b f 0.6 (16)b f
~~
< 3.0nM
1.8 f 0.4 (4) PTH 2.5 nM 1.7 f 0.2 (46) + T 10-13 M 1.5 f 0.1 (46) + M + lo-" M 1.5 f 0.3 (11) + lo-'' M 1.8 f 0.1 (10) + 10-9 M 1.3 f 0.2 (lo) + lo-' M
aPGE, release from unstimulated neonatal mouse calvariae during the first 24 h of culture (preculture) with either high (greater than 6.0 nM) or low (less than 3.0 nM) PGE, concentration in control cultures. Values are means * SEM for N cultures. bSignificant effect of androgen, P < 0.01.
a I n cultured neonatal mouse calvariae after 24 h of P T H treatment following 24 h of preculture with and without T. Values are means + SEM for N cultures. bsignificantly different from P T H alone, P < 0.01.
1185
ANDROGEN EFFECTS ON CULTURED CALVARIAE
nase-digestible protein (CDP) and noncollagen protein (NCP).(43)The percentage collagen synthesis (OroCS) was corrected for the relative abundance of proline in collagen and noncollagen protein. Data from treatment groups of 5-12 calvariae each from two to nine experiments were pooled (except for the time course experiment) and are presented as means f SEM. Statistical significance of differences among means was determined by analysis of variance with post hoc comparison of more than two means by the Bonferrcini method.(44) Unless stated otherwise, differences are considered significant only when P < 0.01.
RESULTS During the 24 h preculture, the effect of androgen on PGE, release from unstimulated calvariae depended on the 0-0 0-0
Control PTH 2.5 nM 0
4 A-AT+PTH
/
/I
TIME (h)
FIG. 1. Effect of T on PTH-stimulated PGE, production in cultured neonatal calvariae as a function of time. Each bone was precultured for 24 h in 1 ml medium with or without lo-' M T and then transferred to similar medium with 2.5 nM PTH. Media were sampled (0.1 ml) at the indicated times. Data were corrected for the media removed. Each point represents the mean f SEM for six bones in one experiment. The effect of T on PTH-stimulated PGE, production was significant ( P < 0.05) at 6, 12, and 24 h.
level of PGE, release in control cultures (Table I). The experiments fell naturally into two groups, with either greater than 6.0 nM or less than 3.0 nM PGE, release from control cultures. When medium PGE, concentration was less than 3.0 nM in the control groups there was no inhibition of PGE, release by T. When medium PGE, concentration was greater than 6 nM in control cultures there were significant 30-50% decreases in PGE, release by T or DHT. At 48 and 72 h of culture, medium PGE, concentration was less than 1.O nM in all unstimulated cultures, and no effect of T or DHT was seen. 4sCa release was unaffected by androgen treatment at any time in unstimulated cultures (data not shown). Treatment of calvariae for 24 h with 2.5 nM PTH increased the release of PGE, by ninefold (Table 2). T at 10-9-10-'2 M inhibited PTH-stimulated PGE, release by 40-50%, but the effects were significant ( P < 0.01) only at lo-" M for the pooled data. In a single experiment looking at the time course of the PGE, response, PTH significantly increased PGE, production at 6 h and this increase was sigM T (Fig. 1). nificantly inhibited by 45Carelease was increased from 9.0 f 0.4 (n = 29) to 19.3 f 0.5% (n = 36) by 48 h of PTH treatment ( P < 0.01). 45Carelease in cultures treated with both PTH and 10-9-10-'2M T for 48 h ranged from 17.0 f 0.7 to 18.4 f 0.7% and was not significantly different from cultures treated with PTH alone. As shown previously,f45t48 h of PTH treatment inhibited both CDP and VoCS. This inhibition was not affected by T (Table 3). A slight reduction in VoCS relative to control was found with lo-' M T but not at other concentrations. Treatment with 0.1 ng/ml of IL-1 for 24 h increased PGE, release 19- to 22-fold, and this stimulated release was decreased 60% by M T and 70-75% by and lo-" M DHT (Table 4). At 48 h stimulated PGE, release was greater and was reduced 75-85% by lo-' and lo-'' M T and 75-95% by 10-9-10-L' DHT (Table 4). No significant inhibition was seen at and lo-" M T or DHT. At 48 h IL-I increased 4sCarelease by 1.6- to 1 %fold, and signifi-
TABLE3. EFFECTS OF TESTOSTERONE (T) ON COLLAGENASE-DIGESTIBLE PROTEIN (CDP), NONCOLLAGEN PROTEIN (NCP), AND % COLLAGEN SYNTHESIS IN CULTURED NEONATAL MOUSE CALVARIAE WITHAND WITHOUT 48 h OF PTH TREATMENT^ CDP
NCP
cs
Treatmen t
N
(dpm/pg dry weight)
(dpm/pg dry weight)
(W
Control T lo-" M T 10-9 M T lo-' M
17 19 20 14
40.4 f 2.3 34.1 f 1.4 34.1 f 1.2 34.9 f 1.4
27.0 26.0 29.3 26.4
PTH 2.5 nM + lo-'" M + T 10-9 M + T lo-* M
20 20 20 14
16.9 f 1.0~ 17.3 0.7~ 18.3 f 0 . 9 ~ 17.9 f 1.w
32.1 f 1.4 33.9 f 1.0 37.0 f 1.2 37.7 f 1.6
*
aValues are means f SEM for N cultures. bSignificanteffect of T, P < 0.01. CSignificanteffect of PTH, P < 0.01.
i
1.4
f
1.1
f
1.4 1.0
f
21.8 f 0.8 19.6 f 0.5 18.1 f 0.8b 19.7 f 0.5 8.9 f 0 . 3 ~ 8.6 f 0 . 2 ~ 8.3 f 0 . 2 ~ 8.1 f 0 . 3 ~
1186
PILBEAM AND RAISZ TABLE4. EFFECTSOF T OR DHT ON PGE, RELEASE FROM NEONATAI MOUSECALVARIAE CULTURED FOR 24 OR 48 h WITH RECOMBINANT WITH HUMANIL-la (IL-1) AFTER24 h OF PRECULTURE AND WITHOUT T OR DHTa ~~
Medium PCE, concentration, nM (N) Treatment
24 h
48 h
Control IL-1 0.1 ng/ml +T M + T 10'" M +T M + T 10-9 M
0.5 f 0.1 (24)b 10.9 f 1.5 (32) 7.4 f 1.5 (15) 6.1 f 1.3 (19) 4.3 f 1.0 (18)b 5.0 f 1.2 (13)
0.1 f 0.0 (24)b 19.4 f 3.3 (34) 6.3 f 1.9 (13) 10.2 f 3.2 (20) 3.2 f 1.0 (18)b 4.4 f 1.0 (13)b
Control IL-1 0.1 ng/ml + DHT M + DHT lo-'' M + DHT M + DHT 1 0 - ~M
0.9 f 0.2 (10)b 16.7 f 2.4 (21) 8.5 f 1.4 (8) 4.3 f 0.8 (12)b 5.0 f 0.7 (I2)b 9.0 f 1.6 (8)
0.3 f 0.2 (1l)b 21.2 f 4.5 (20) 4.8 f 2.2 (7) 0.6 f 0.2 (12)b 4.3 f 1.3 (12)b 2.3 f 1.0 (7)b
____
aValues are means f SEM for N cultures. bsignificantly different from IL-l alone, P < 0.01.
cant reductions were seen with lo-" M T (from 15.5 + 0.6 to 12.5 f 0.5%, P < 0.05) and with lo-'' M DHT (from 15.3 + 0.7 to 11.2 + 1.1%, P < 0.01).
DISCUSSION Recently we reported that oophorectomy increased and 17@-estradiol(BE2) treatment in vivo decreased PGE, production by calvariae excised from 7-week-old rats, but we were unable to show a direct effect of PE, on these calvariae in v i t r ~ . ( . "We ~ subsequently developed a culture system, using calvariae from neonatal mice, in which we demonstrated that PTH-stimulated PGE, production could be inhibited 50-70% by PE, at concentrations of 10'9-10-'2 M and that PTH-stimulated 45Carelease could also be inhibited by about 20% with PE, at 1 0 - l l - l P z M.(401We have also found that physiologic concentrations of @E2can inhibit IL- 1-stimulated PGE, production. i 4 b ) In the present study, using the same culture system, we have now been able to show a direct effect of T and DHT on bone. Specifically, we have shown that T inhibits PGE, production in unstimulated cultures with high control PGE, release and in PTH- or IL-1-stimulated cultures but not in unstimulated cultures with low PGE, production. The maximum inhibitory effect ranged from 40 to 50% in unstimulated and PTH-stimulated cultures to more than 80% in IL-1-stimulated cultures. The variable production of PGE, in control cultures could be due to differences in the endogenous production of cytokines, such as IL-1 or TGF-fi, which can stimulate PGE, release. Dose-response curves for the effects of T and DHT on PGE, production, although quite variable, were generally biphasic, with
maximal effects at 10-lo-lO'll M . A similar biphasic response was also seen in our previous study of the PE, inhibition of PTH-stimulated PGE, DHT had an inhibitory effect similar to or greater than T in unstimulated or IL-I-stimulated cultures. Hence, the inhibitory effect of T is probably not due to conversion of T to estrogen. We found no effect of T on resorption as measured by 45Carelease in unstimulated cultures, and the effects of T on PTH-stimulated 4sCa release were not significant. In our previous study we found small but significant decreases in PTH-stimulated 45Carelease with PE, that appeared to be independent of PGE, production.(40'In this study there were small but significant reductions of 2030% in IL-1-stimulated 45Carelease with both T and DHT. The changes in IL-1-stimulated 45Carelease did not parallel changes in PGE, production. This finding is consistent with previous studies showing that the resorptive effect of IL-1 is variably related to PGE, T did not increase [JH]Similar to previous proline incorporation into collagen or reduce the PTH inhibition of collagen synthesis. The lack of stimulation of CDP by T in short-term cultures does not rule out longer term effects of changes in prostaglandin production on bone turnover in vitro or in vivo. It is not yet possible to assess the importance of these observations in explaining the known effects of androgens in vivo, but if physiologic concentrations of T and DHT regulate prostaglandin production in vivo, then increased prostaglandin production might be expected in hypogonadal men and could contribute to increased bone turnover. The similarity of the effects of estrogen and androgen in our culture system is consistent with in vivo observations since both sex hormones are able to decrease bone turnover.
1187
ANDROGEN EFFECTS ON CULTURED CALVARIAE
ACKNOWLEDGMENTS This work was supported by NIH Grants AM-18063 and AR-38933. Carol Pilbeam is supported by a Brookdale National Fellowship Award in Geriatrics. A preliminary report of this work was presented at the combined meetings of the American Society for Bone and Mineral Research and the International Conference on Calcium Regulating Hormones, Montreal, Canada, September 1989.
16.
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1188 lagen synthesis in vitro. Calcif Tissue Res 25105-1 10. 34. Caputo CB, Meadows D, Raisz LG 1976 Failure of estrogens and androgens to inhibit bone resorption in tissue culture. Endocrinology 98: 1065- 1068. 35. Gray TK, Mohan S, Linkhart TA, Baylink DJ 1989 Estradiol stimulates in vitro the secretion of insulin-like growth factors by the clonal osteoblastic cell line, UMR-106. Biochem Biophys Res Commun 158:407-412. 36. Pacifici R, Rifas L, Teitelbaum S, Slatopolsky E, McCracken R, Bergfeld M, Lee W, Avioli LV, Peck WA 1987 Spontaneous release of interleukin-I from human blood monocytes reflects bone formation in idiopathic osteoporosis. Proc Natl Acad Sci USA 844616-4620. 37. Pacifici R, Rifas L, McCracken R, Vered I, McMurtry C, Avioli LV, Peck WA 1989 Ovarian steroid treatment blocks a postmenopausal increase in blood monocyte interleukin-I release. Proc Natl Acad Sci USA 86:2389-2402. 38. Axelrod DW, Lachman LB, Judge D, Mallette LE, Gagel RF 1989 Resorptive hypercalciuria and increased interleukin 1 in a young man with hypogonadism and osteoporosis: Reversal with androgen treatment (abstract). Clin Res 37:355A. 39. Feyen JHM, Raisz LG 1987 Prostaglandin production by calvariae from sham operated and oophorectomized rats: Effects of 170-estradiol in vivo. Endocrinology 121:819-821. 40. Pilbeam CC, Klein-Nulend J, Raisz LG 1989 Inhibition by 170-estradiol of PTH stimulated resorption and prostaglandin production in cultured neonatal mouse calvariae. Biochem Biophys Res Commun 163:1319-1324. 41. Raisz LG, Simmons HA 1985 Effects of parathyroid hormone and cortisol on prostaglandin production by neonatal
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Address reprint requests to: Dr. Carol Pilbeam Division of Endocrinology/Departtnent of Medicine University of Connecticut Health Center Farmington, CT 06032 Received for publication April 19, 1990; in revised form May 30, 1990; accepted June 7, 1990.
Effects of Androgens on Parathyroid Hormone and Interleukin-1-Stimulated Prostaglandin Production in Cultured Neonatal Mouse Calvariae CAROL C. PILBEAM and LAWRENCE G . RAISZ
ABSTRACT In this study we show direct inhibitory effects on prostaglandin E2 (PGE,) production by the androgens, testosterone (T) and dihydrotestosterone (DHT), in cultured neonatal mouse calvariae. After 24 h of preculture with or without androgens, bones were treated with bovine (&34)-parathyroid hormone (PTH) or recombinant human interleukin-la (IL-1). During preculture androgens decreased PGE, release only in those experiments in which control PGE, was high. PTH increased medium PGE, 9-fold at 24 h, and lo-" M T inhibited this increase by 50%. Treatment with IL-1 for 24 h increased medium PGE, 19- to 22-fold, and lo-'' M T and DHT inhibited this increase by 60 and 70'70, respectively. T did not significantly affect the PTH-stimulated release of previously incorporated 4sCaor alter the PTH inhibition of incorporation of [JH]prolineinto collagenase-digestible protein. IL-1 stimulated 45Carelease by 60-80%, and small but significant reductions of 20-30070 were seen with T and DHT. This study shows that T and DHT have direct effects on bone at physiologic concentrations, similar to our previous study in which PTH-stimulated PGE, production in the same culture system was inhibited by physiologic concentrations of 17@-estradiol,and suggests that prostaglandins may mediate some of the effects of androgens in vivo.
INTRODUCTION are potent regulators of bone metabolism in vivo. Deficiency of either is associated with reduced bone mass and increased risk for osteoporosis in both humans and Men with hypogonadism secondary to an isolated deficiency of hypothalamic gonadotropin releasing hormone have decreased cortical and spinal trabecular bone density compared to normal individualsI6) and have shown increases in bone density during gonadal steroid treatment.I7) As in women after estrogen w i t h d r a ~ a l , ' ~androgen .~) deficiency may be associated with increased bone resorption and loss of bone mass compared to normal controls, ID) and the increased turnover may be reduced by androgen replacement . ( I 1 ) However, other studies have suggested that the primary abnormality associated with hypogonadism in men may be
A
NDROGENS AND ESTROGENS
impaired bone formation.(b' " 1 3 ) Androgens also influence bone metabolism in women as well as in men,(I41 5 ) and anabolic steroids have been used to treat osteoporotic women.('b 1 7 ) The mechanism by which androgens and estrogens influence bone metabolism in vivo is unknown. Estrogen has been shown to inhibit parathyroid hormone (PTH)-stimulated bone resorption in postmenopausal women with primary hyperparathyroidism.(18-'0)An in vivo study suggested increased sensitivity of bone to PTH in castrated male mice. [ ' I ) Similar to other androgen target tissues, human bone is capable of metabolizing testosterone (T) to dihydrotestosterone (DHT).IZ2)Direct effects of sex hormones on bone metabolism have been suggested by recent studies that have demonstrated both estrogen and androgen receptors in cultured human osteoblastlike cells from both males and females(z3 and in human osteosarcoma cells."' z6)
Division of Endocrinology and Metabolism, Department of Medicine, University of Connecticut Health Center, Farmington, C T 06032.
1183
PILBEAM AND RAISZ
1184
Both estrogen and androgen have been shown to affect cell proliferation and alkaline phosphatase production in osteoblastlike cell^.'^'-^'' Both have also been shown to inand to decrease PTHcrease type I collagen mRNA(25~'6~30) stimulated CAMP p r o d ~ c t i o n ( ~ *in~ ~human ) osteosarcoma cells. However, attempts to show a direct effect of androgen at physiologic concentrations on bone collagen synthesisi33'and on PTH-stimulated bone in organ culture have been unsuccessful. Several studies have suggested that the actions of androgens and estrogens may be mediated by autocrine-paracrine factors. Studies in osteoblastlike cells have shown that estrogen can increase secretion of insulinlike growth and mRNA for insulinlike growth factor-I (IGFI)"') and that both androgen and estrogen can increase In mRNA for transforming growth factor /3 (TGF-fi).i25.26) vivo studies have related sex steroid deficiency to increased interleukin-l (IL-1) production by monocytes in both men and ~ o m e n . ( ~ ~We - ' ~have ) shown that oophorectomy increased prostaglandin E2 (PGE,) production by excised rat ~alvariae'~') and that estrogen treatment in vitro reduced PTH-stimulated PGE, production by neonatal mouse cal~ a r i a e . ' ~ In ' ) the present study we demonstrate a direct effect of physiologic concentrations of T and DHT on PTHand IL-I-stimulated PGE, production and on IL-I-stimulated resorption in cultured neonatal mouse calvariae.
MATERIALS AND METHODS Timed pregnant CDI mice (Charles River, Wilmington, MA) were injected on day 16 of gestation with 50 pCi of Y a (New England Nuclear, Boston, MA) to label fetal bones in utero. Calvariae were excised from the neonates 7 days after birth, and pieces of parietal bone about 2 x 3 mm were dissected free of all sutures. Bones were incubated in 12-well plates (Costar, Cambridge, MA) in 1 ml medium on a rocker platform at 37°C in a humidified atmosphere of 5% CO, in air. Bones from each litter were
TABLE1. EFFECTS OF TESTOSTERONE (T) OR DIHYDROTESTOSTERONE (DHT) ON PROSTAGLANDIN E, (PGE,) RELEASE^
distributed equally throughout the different treatment groups. All experiments had stimulated and unstimulated control groups, but not all androgen doses were in every experiment. The basic culture medium was phenol red-free BGJb (GIBCO, Grand Island, NY) with 1 mg/ml of bovine serum albumin (BSA, radioimmunoassay grade, Sigma, St. Louis, MO), 1 mM proline (Sigma), 0.4 mM 1glutamine (Sigma), 0.6 mM I-ascorbic acid (Sigma), and 3 mM phosphate. Bones were precultured for 24 h with or without T (Sigma) or DHT (Sigma) and then transferred to similar media with or without 2.5 nM synthetic bovine (1-34)-PTH (Bachem, Torrance, CA) or 0.1 ng/ml of recombinant human 1L-la (kindly provided by Dr. P. Lomedico, Hoffmann-LaRoche, Nutley, NJ) and cultured for another 48 h with a medium change after 24 h. Androgens were dissolved in ethanol, and media for all treatment and control groups contained 0.1070 ethanol. Medium was assayed at the end of preculture and at 24 and 48 h after the addition of PTH or IL-I for PGE, using radioimmunoassay (RIA, lower limit of detection 0.10 nM), as described previously. ( 4 1 ) PGE, antibodies were kindly provided by Dr. N.H.L. Hunt, Canberra, Australia. To prevent the interference of W a with 3H in the RIA, medium calcium was precipitated before RIA with ammonium oxalate (1 pl saturated solution added to 20 p1 medium, incubated 1 h at 37"C, and centrifuged at 1500 cpm and 4°C for 15 minutes). The W a content of the medium at 24 and 48 h after addition of PTH or IL-1 and of the 5 % (v/v) trichloroacetic acid (TCA) extracts of the bones was measured by liquid scintillation counting and used to calculate the total W a and the cumulative percentage of "'Ca released, as described previously.(42) To assess collagen and noncollagen protein synthesis, bones were pulsed with 5 pCi/ml of [3H]proline (New England Nuclear) for the last 2 h of a 48 h culture. Bones were extracted with 5% TCA, acetone, and ether, dried, weighed, homogenized, and treated with purified bacterial collagenase (Worthington Biochemical Corp., Freehold, NJ) to measure incorporation of [3H]proline in collage-
OF TESTOSTERONE (T) ON MEDIUM TABLE2. EFFECTS PROSTAGLANDIN E, (PGE,) CONCENTRATION^
Treatment
N
Medium PGE, concentration fnW
Control
35 42 11 24 24 19 18 14
0.5 f 0.lb 4.6 f 0.5 3.3 f 1.0 2.6 f 0.5 2.2 f 0.3b 2.8 f 0.4 2.5 f 0.3 3.9 f 0.7
Medium PGE, concentration, nM (N)
> 6.0nM
Treatment
Control
Control T lo-" M T lo-'' M
7.3 4.1 4.9
Control DHT lo-" M DHT lo-" M
7.4 f 0.4 (44) 3.6 f 0.2 (24)b 3.9 f 0.3 (24)b
Control
0.4 (55) f 0.3 (20)b f 0.6 (16)b f
~~
< 3.0nM
1.8 f 0.4 (4) PTH 2.5 nM 1.7 f 0.2 (46) + T 10-13 M 1.5 f 0.1 (46) + M + lo-" M 1.5 f 0.3 (11) + lo-'' M 1.8 f 0.1 (10) + 10-9 M 1.3 f 0.2 (lo) + lo-' M
aPGE, release from unstimulated neonatal mouse calvariae during the first 24 h of culture (preculture) with either high (greater than 6.0 nM) or low (less than 3.0 nM) PGE, concentration in control cultures. Values are means * SEM for N cultures. bSignificant effect of androgen, P < 0.01.
a I n cultured neonatal mouse calvariae after 24 h of P T H treatment following 24 h of preculture with and without T. Values are means + SEM for N cultures. bsignificantly different from P T H alone, P < 0.01.
1185
ANDROGEN EFFECTS ON CULTURED CALVARIAE
nase-digestible protein (CDP) and noncollagen protein (NCP).(43)The percentage collagen synthesis (OroCS) was corrected for the relative abundance of proline in collagen and noncollagen protein. Data from treatment groups of 5-12 calvariae each from two to nine experiments were pooled (except for the time course experiment) and are presented as means f SEM. Statistical significance of differences among means was determined by analysis of variance with post hoc comparison of more than two means by the Bonferrcini method.(44) Unless stated otherwise, differences are considered significant only when P < 0.01.
RESULTS During the 24 h preculture, the effect of androgen on PGE, release from unstimulated calvariae depended on the 0-0 0-0
Control PTH 2.5 nM 0
4 A-AT+PTH
/
/I
TIME (h)
FIG. 1. Effect of T on PTH-stimulated PGE, production in cultured neonatal calvariae as a function of time. Each bone was precultured for 24 h in 1 ml medium with or without lo-' M T and then transferred to similar medium with 2.5 nM PTH. Media were sampled (0.1 ml) at the indicated times. Data were corrected for the media removed. Each point represents the mean f SEM for six bones in one experiment. The effect of T on PTH-stimulated PGE, production was significant ( P < 0.05) at 6, 12, and 24 h.
level of PGE, release in control cultures (Table I). The experiments fell naturally into two groups, with either greater than 6.0 nM or less than 3.0 nM PGE, release from control cultures. When medium PGE, concentration was less than 3.0 nM in the control groups there was no inhibition of PGE, release by T. When medium PGE, concentration was greater than 6 nM in control cultures there were significant 30-50% decreases in PGE, release by T or DHT. At 48 and 72 h of culture, medium PGE, concentration was less than 1.O nM in all unstimulated cultures, and no effect of T or DHT was seen. 4sCa release was unaffected by androgen treatment at any time in unstimulated cultures (data not shown). Treatment of calvariae for 24 h with 2.5 nM PTH increased the release of PGE, by ninefold (Table 2). T at 10-9-10-'2 M inhibited PTH-stimulated PGE, release by 40-50%, but the effects were significant ( P < 0.01) only at lo-" M for the pooled data. In a single experiment looking at the time course of the PGE, response, PTH significantly increased PGE, production at 6 h and this increase was sigM T (Fig. 1). nificantly inhibited by 45Carelease was increased from 9.0 f 0.4 (n = 29) to 19.3 f 0.5% (n = 36) by 48 h of PTH treatment ( P < 0.01). 45Carelease in cultures treated with both PTH and 10-9-10-'2M T for 48 h ranged from 17.0 f 0.7 to 18.4 f 0.7% and was not significantly different from cultures treated with PTH alone. As shown previously,f45t48 h of PTH treatment inhibited both CDP and VoCS. This inhibition was not affected by T (Table 3). A slight reduction in VoCS relative to control was found with lo-' M T but not at other concentrations. Treatment with 0.1 ng/ml of IL-1 for 24 h increased PGE, release 19- to 22-fold, and this stimulated release was decreased 60% by M T and 70-75% by and lo-" M DHT (Table 4). At 48 h stimulated PGE, release was greater and was reduced 75-85% by lo-' and lo-'' M T and 75-95% by 10-9-10-L' DHT (Table 4). No significant inhibition was seen at and lo-" M T or DHT. At 48 h IL-I increased 4sCarelease by 1.6- to 1 %fold, and signifi-
TABLE3. EFFECTS OF TESTOSTERONE (T) ON COLLAGENASE-DIGESTIBLE PROTEIN (CDP), NONCOLLAGEN PROTEIN (NCP), AND % COLLAGEN SYNTHESIS IN CULTURED NEONATAL MOUSE CALVARIAE WITHAND WITHOUT 48 h OF PTH TREATMENT^ CDP
NCP
cs
Treatmen t
N
(dpm/pg dry weight)
(dpm/pg dry weight)
(W
Control T lo-" M T 10-9 M T lo-' M
17 19 20 14
40.4 f 2.3 34.1 f 1.4 34.1 f 1.2 34.9 f 1.4
27.0 26.0 29.3 26.4
PTH 2.5 nM + lo-'" M + T 10-9 M + T lo-* M
20 20 20 14
16.9 f 1.0~ 17.3 0.7~ 18.3 f 0 . 9 ~ 17.9 f 1.w
32.1 f 1.4 33.9 f 1.0 37.0 f 1.2 37.7 f 1.6
*
aValues are means f SEM for N cultures. bSignificanteffect of T, P < 0.01. CSignificanteffect of PTH, P < 0.01.
i
1.4
f
1.1
f
1.4 1.0
f
21.8 f 0.8 19.6 f 0.5 18.1 f 0.8b 19.7 f 0.5 8.9 f 0 . 3 ~ 8.6 f 0 . 2 ~ 8.3 f 0 . 2 ~ 8.1 f 0 . 3 ~
1186
PILBEAM AND RAISZ TABLE4. EFFECTSOF T OR DHT ON PGE, RELEASE FROM NEONATAI MOUSECALVARIAE CULTURED FOR 24 OR 48 h WITH RECOMBINANT WITH HUMANIL-la (IL-1) AFTER24 h OF PRECULTURE AND WITHOUT T OR DHTa ~~
Medium PCE, concentration, nM (N) Treatment
24 h
48 h
Control IL-1 0.1 ng/ml +T M + T 10'" M +T M + T 10-9 M
0.5 f 0.1 (24)b 10.9 f 1.5 (32) 7.4 f 1.5 (15) 6.1 f 1.3 (19) 4.3 f 1.0 (18)b 5.0 f 1.2 (13)
0.1 f 0.0 (24)b 19.4 f 3.3 (34) 6.3 f 1.9 (13) 10.2 f 3.2 (20) 3.2 f 1.0 (18)b 4.4 f 1.0 (13)b
Control IL-1 0.1 ng/ml + DHT M + DHT lo-'' M + DHT M + DHT 1 0 - ~M
0.9 f 0.2 (10)b 16.7 f 2.4 (21) 8.5 f 1.4 (8) 4.3 f 0.8 (12)b 5.0 f 0.7 (I2)b 9.0 f 1.6 (8)
0.3 f 0.2 (1l)b 21.2 f 4.5 (20) 4.8 f 2.2 (7) 0.6 f 0.2 (12)b 4.3 f 1.3 (12)b 2.3 f 1.0 (7)b
____
aValues are means f SEM for N cultures. bsignificantly different from IL-l alone, P < 0.01.
cant reductions were seen with lo-" M T (from 15.5 + 0.6 to 12.5 f 0.5%, P < 0.05) and with lo-'' M DHT (from 15.3 + 0.7 to 11.2 + 1.1%, P < 0.01).
DISCUSSION Recently we reported that oophorectomy increased and 17@-estradiol(BE2) treatment in vivo decreased PGE, production by calvariae excised from 7-week-old rats, but we were unable to show a direct effect of PE, on these calvariae in v i t r ~ . ( . "We ~ subsequently developed a culture system, using calvariae from neonatal mice, in which we demonstrated that PTH-stimulated PGE, production could be inhibited 50-70% by PE, at concentrations of 10'9-10-'2 M and that PTH-stimulated 45Carelease could also be inhibited by about 20% with PE, at 1 0 - l l - l P z M.(401We have also found that physiologic concentrations of @E2can inhibit IL- 1-stimulated PGE, production. i 4 b ) In the present study, using the same culture system, we have now been able to show a direct effect of T and DHT on bone. Specifically, we have shown that T inhibits PGE, production in unstimulated cultures with high control PGE, release and in PTH- or IL-1-stimulated cultures but not in unstimulated cultures with low PGE, production. The maximum inhibitory effect ranged from 40 to 50% in unstimulated and PTH-stimulated cultures to more than 80% in IL-1-stimulated cultures. The variable production of PGE, in control cultures could be due to differences in the endogenous production of cytokines, such as IL-1 or TGF-fi, which can stimulate PGE, release. Dose-response curves for the effects of T and DHT on PGE, production, although quite variable, were generally biphasic, with
maximal effects at 10-lo-lO'll M . A similar biphasic response was also seen in our previous study of the PE, inhibition of PTH-stimulated PGE, DHT had an inhibitory effect similar to or greater than T in unstimulated or IL-I-stimulated cultures. Hence, the inhibitory effect of T is probably not due to conversion of T to estrogen. We found no effect of T on resorption as measured by 45Carelease in unstimulated cultures, and the effects of T on PTH-stimulated 4sCa release were not significant. In our previous study we found small but significant decreases in PTH-stimulated 45Carelease with PE, that appeared to be independent of PGE, production.(40'In this study there were small but significant reductions of 2030% in IL-1-stimulated 45Carelease with both T and DHT. The changes in IL-1-stimulated 45Carelease did not parallel changes in PGE, production. This finding is consistent with previous studies showing that the resorptive effect of IL-1 is variably related to PGE, T did not increase [JH]Similar to previous proline incorporation into collagen or reduce the PTH inhibition of collagen synthesis. The lack of stimulation of CDP by T in short-term cultures does not rule out longer term effects of changes in prostaglandin production on bone turnover in vitro or in vivo. It is not yet possible to assess the importance of these observations in explaining the known effects of androgens in vivo, but if physiologic concentrations of T and DHT regulate prostaglandin production in vivo, then increased prostaglandin production might be expected in hypogonadal men and could contribute to increased bone turnover. The similarity of the effects of estrogen and androgen in our culture system is consistent with in vivo observations since both sex hormones are able to decrease bone turnover.
1187
ANDROGEN EFFECTS ON CULTURED CALVARIAE
ACKNOWLEDGMENTS This work was supported by NIH Grants AM-18063 and AR-38933. Carol Pilbeam is supported by a Brookdale National Fellowship Award in Geriatrics. A preliminary report of this work was presented at the combined meetings of the American Society for Bone and Mineral Research and the International Conference on Calcium Regulating Hormones, Montreal, Canada, September 1989.
16.
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Address reprint requests to: Dr. Carol Pilbeam Division of Endocrinology/Departtnent of Medicine University of Connecticut Health Center Farmington, CT 06032 Received for publication April 19, 1990; in revised form May 30, 1990; accepted June 7, 1990.
