- Email: [email protected]
The Effects of Androgens on Osteoblast Function In Vitro
Symposium Article
The Effects of Androgens on Osteoblast Function In Vitro SUNDEEP KHOSLA,
MD
The relative contributions of androgens and estrogen to bone metabolism are still being defined. To directly assess androgen effects on bone cells, we have developed human osteoblastic cell models expressing the androgen receptor. This approach is distinct from studies that have
used a variety of transformed cell lines, in that the latter studies showed variability in the results. Investigation into androgen effects on bone has been overshadowed by interest in estrogen effects. Mayo Clin Proc. 2000;75(suppl):S51-S54
T
he presence of the estrogen receptor in human bone cells was described more than a decade ago by Eriksen Ind colleagues. I Their work was followed by numerous ~dies on estrogen's effects in vitro on bone cells. A few years later the presence of the androgen receptor ~ bone cells was described by the same group- and was ~ter confirmed by others. Remarkably, this finding was ~t followed by a comparable explosion in studies of ~drogen's effects on bone cells. Perhaps because osteo~rosis is predominantly a women's disease, the study of ~trogen's effects was emphasized instead. Osteoporosis Mso is a disease of men, however, and the effects of andro~ens on bone in either sex have not been studied to the e degree as that of estrogen. Figure 1 depicts the relative levels of estrogen and drogen receptors in human bone cells by sex, expressed molecules per bone cell nucleus. Data from subsequent ~lUdies by others are listed in Table 1. Values are expressed ~or the androgen receptor per cell in primary human ~steoblasts and also for a variety of human osteosarcoma lines. When the effects of androgens on osteoblast prolif~ation were studied, androgens were generally found to [enhance proliferation (Table 2). There are relatively sig~ficant effects on osteoblast proliferation in general and ~so specifically with dihydrotestosterone (DHT) and ldehydroepiandrosterone (DHEA).
therefore, much of this variability could be attributed to methodologic differences. In an effort to explain the variability, it was noted that studies done in primary human osteoblasts, in fact, used bone chips in culture and then examined whatever grew out.' The result-a mixture of fibroblastic cells and osteoblastic cells-demonstrated variable percentages of alkaline phosphatase staining. This raises the question of whether osteoblast-related effects or effects due to other cell types at varying stages of differentiation were being studied. Similar variability arose in systems using rat cells. In cases when osteosarcoma-TE85 cells-had been used, there appeared to be a decrease in proliferation.' So these various systems added controversy te the observations being made about androgen effects on proliferation. If one looks at differentiation markers, namely, alkaline phosphatase or type I collagen, osteocalcin, or mineralization of extracellular matrix, the results have, in general, shown "anabolic" effects of androgen, although these are variable (Table 3). In studies done on primary cultures from bone chips, it was unclear what cell population was being studied. Nonetheless, in general, one sees an increase in the percentage of cells expressing alkaline phosphatase, either an increase or no change in type I collagen and proline incorporation, and either an increase or no change in osteocalcin secretion.> Various growth factors, or cytokines, such as transforming growth factor ~ (TGF-~), also have been studied (Table 4). Androgens, including testosterone, DHT, and DHEA, increased mRNA levels and activity of a number of growth factors.w? The results also showed that the insulin-like growth factor II (IGF-II) receptor was increased by DHT.6 Interleukin 1~ (IL-l~) activity also was increased with androgen stimulation, which is surprising, since, in the reported osteoblastic systems, estrogen has tended to decrease IL-l ~ production.f IL-6 was studied in a preadipocytic marrow cell line, where it appeared that pro-
~
SUMMARY OF ANDROGEN EFFECTS DN BONE CELLS Some of the apparent variability in androgen receptor values probably arises from the methods used to determine the number of binding sites, ie, whether an antibody or a 'specific nuclear binding assay was used. Theoretically, From the Division of Endocrinology, Metabolism, Nutrition, and Internal Medicine, Mayo Clinic Rochester, Rochester, Minn. Presented at Symposium on Testosterone Replacement in Elderly Men, Palm Springs, Calif, March 18-20, 1999. Mayo Clin Proc. 2000;75(suppl):S51-S54
S51
© 2000 Mayo Foundation for Medical Education and Research
The Effects of Androgens on Osteoblast Function In Vitro
S52
4,000
3.000
Moleculesl nucleus 2,000
1,000
Estrogen receptor
a a
••
a
••
~
0
o~o Male
• •
••
•• •• Female
Table 2.-Effects of Androgens on Osteoblast Proliferati«m Cells
•••
a a
a
Androgen receptor
Mayo Clin Proc, January 2000, Vol 75 (Supplement)
0
Ga 000
808 Male
••• ~
•• •
Androgen(s)
Effect
Primary human osteoblasts Primary human osteoblasts
DHT DHT DHEA
t50% t200% t88%
Primary rat diaphyseal osteoblasts Primary rat epiphyseal cells Primary rat calvarial osteoblasts
T T DHT T
tlOO% t57% t 150% t70%
TE-85 osteosarcoma cells
DHT T
.25% .20%
Female
*Adapted from Hofbauer and Khosla.f Figure 1. Relative levels of estrogen and androgen receptors by gender in human bone cells. Adapted from Eriksen et all and Colvard et al.2
duction was decreased by androgens." Prostaglandin Eb another potential mediator of bone resorption with sex steroid deficiency, was decreased by androgens. 10 While the literature contains data pertaining to androgen effects on bone, these effects have not been as well studied as those of estrogen. The data are sparse and are confounded by differences in the model systems that generated them. One can characterize the sources of variability in the data as arising from differences in donor age (fetal, neonatal, adolescent, adult), skeletal site (vertebral, femoral, calvarial), type of bone (cancellous, cortical), species, degree of transformation (osteosarcoma cell lines vs conditionally immortalized cells vs primary cultures), and stage of differentiation of the cells. Variability in species can present important differences, particularly with respect to growth factors. In human bone, the predominant IGF involved in bone metabolism is IGF-II, whereas in rat bone it is IGF-I. Many studies use rat bone. The degree of cellular transformation is another impor-
Table I.-Androgen Receptors in Primary Human Osteoblasts and Human Osteosarcoma Cell Lines* Cells
Characteristics
hOB hOB hOB
Primary human osteoblasts Primary human osteoblasts Primary human osteoblasts
146 ± 19 821 ± 140 5520 ± 240
TE85 U2-0S SaOS-2
Human osteosarcoma cell line Human osteosarcoma cell line Human osteosarcoma cell line
2800-3600 1600-3600 1300-5800
*Adapted from Hofbauer and
Khosla. 5
ARicell
tant variable. Many studies have used osteosarcoma cell lines containing multiple genetic abnormalities. Finally, the stage of differentiation of the osteoblasts has often not been considered. The effects of sex steroids on the mature fully differentiated cell vs the osteoblast precursor cell may be quite different. ' DEVELOPING NEW MODEL SYSTEMS To circumvent some of these problems, several years ago our group developed conditionally immortalized cells derived from a human fetus, known as human fetal osteoblast (hFOB) cells. [1 These cells are immortalized with a temperature-sensitive SV40 large T antigen. At the permissive temperature of 34°C, the T antigen is active, and the cells proliferate; however, because of the temperature-sensitive mutation in the T antigen, the cells decrease proliferating when the ambient temperature reaches 39°C.
Table 3.-Androgen Effects on Osteoblast Differentiation Markers* Marker
Androgen(s)
Alkaline phosphatase (AP) Percentage of AP-positive cells AP activity
DHT DHT,DHEA DHT,T
Type 1 collagen mRNAlevels
DHT,T
(3H)proline incorporation
DHT DHT
Osteocalcin secretion
DHT, T,DHEA DHT, T, DHEA
Mineralization of extracellular matrix
DHT, DHEA
*Adapted from Hofbauer and Khosla.>
Change
.. .. .. .. t
t
t
t t
t
\tayo Clin Proc, January 2000, Vol 75 (Supplement)
The Effects of Androgens on Osteoblast Function In Vitro
Table 4.-Androgen Effects on Growth Factors and
Cytokines*
Androgen(s)
Change
TGF-~
DHT,T DHT, T,DHEA
IGF-IIR
DHT
lL-l~
T
t t t t t
1L-6
DHT, T,DHEA DHT,T
....
PGE2
DHT,T
Cytokine
..
Method mRNAlevels Activity mRNA levels Affinity for IGF-II mRNA levels, protein production Reporter construct mRNA levels, protein production Protein production
*Adapted from Hofbauer and Khosla. 5
They then differentiate and express the complete osteoblastic phenotype, including the formation of mineralized nodules. Even though the primary bone cultures for these cells contained estrogen receptors and androgen receptors, perhaps due to continuous culture in vitro, hFOB cells express low levels of both (200 to 300 receptors per nucleus). This is a limitation, as more robust receptor expression is preferable because it leads to good responses, especially in initial studies. While researchers were searching for a more robust response, another cell line was developed-the hFOBI AR cell line.t? The ideal system comprises primary cultures; however, such cultures are difficult to obtain, they proliferate slowly, and they reflect a great deal of interindividual variability, making data interpretation difficult. They also tend toward senescence with multiple passages, which becomes associated with low levels of sex steroid receptors. By using the "conditionally" immortalized cells grown at the restrictive temperature, we at least have a well-defined clonal population of cells with fully understood characteristics to use for beginning examination of factors regulated by androgens in these cells. The expression plasmid used for developing the hFOBI AR cells used the human androgen receptor (hAR) cloned downstream of the cytomegalovirus promoter.There is an SV40 polyadenylation signal downstream of the hAR. Since FOB cells are already gentamicin resistant due to the introduction of the T antigen and the plasmid that encodes it, a different antibiotic resistance was needed. This plasmid has a hygromycin-resistant gene that allows us to alternate antibiotic selection and maintain both the T antigen and the androgen receptor in continuous culture.
S53
STUDIES WITH THE HFOB/AR CELL LINE AND ITS CLONES In initial studies, the parent hFOB cell line had very low but detectable levels of androgen receptors. Several hFOB/AR clones were not very different, but the clone identified as hFOB/AR-6 had much higher levels, which actually increased when DHT was added. In a chloramphenical acetyl-transferase (CAT), a stimulation of computed tomographic activity was shown going from the absence to the presence of androgen. Some data in the literature suggest that there may be important differences between bone and reproductive tissues in terms of androgen autoregulation of the receptor. In prostate cells, androgen decreases androgen receptor levels, whereas the opposite appears to be the case in bone cells.U It would be quite interesting to further explore these regulatory differences in prostate and bone. Nuclear binding studies revealed that the hFOB/AR cells have approximately 3800 receptors. This has been demonstrated in 2 clones, whereas the parent hFOB cell line expressed about 200 to 300 receptors per cell. This model system, while not ideal, could at least provide clues as to which target genes are being regulated by androgens. We have used hFOB/AR cells to study the effects of androgens on IL-6 production.!! These studies indicate that gonadal but not adrenal androgens markedly reduce basal and cytokine-stirnulated IL-6 production by these cells. We also have examined the regulation of the IOF system in these cells. 15 Thus, gonadal, but not adrenal, androgens increase the production of IOF-I by these cells and also have important effects on the IOF-binding proteins. Further studies with these cells involve using differential display and gene-chip technology in attempting to identify other potential androgen-regulated genes in bone cells. CONCLUSIONS Bone cells are clearly androgen responsive, as seen in both primary bone cell cultures and model systems. Variability in results obtained from in vitro studies is largely due to differences in the model systems used. Within model systems, it is necessary to clearly define and control the sources of such variability (ie, donor and bone type, degree of transformation, and stage of differentiation) and to avoid extrapolation from one species to the other. New model systems, such as the hFOB/AR cells and the comparablehFOBIERcells, are useful for identifying the target genes that are regulatedand for understanding the processes that are involved. However,it is necessary to return to the primary nontransfected systems to verify the results.
S54
The Effects of Androgens on Osteoblast Function In Vitro
ACKNOWLEDGMENTS The work of Francesca Gori, Lawrence Hofbauer, Kevin Hickock, and Cheryl Conover is gratefully acknowledged, as is the contribution of Dr Neary at Schering-Plough.
REFERENCES 1.
2. 3. 4.
5. 6. 7.
Eriksen EF, Colvard DS, Berg NJ, et al. Evidence of estrogen receptors in normal human osteoblast-like cells. Science. 1988; 241:84-86. Colvard DS, Eriksen EF, Keeting PE, et al. Identification of androgen receptors in normal human osteoblast-like cells. Proc Natl Acad Sci USA. 1989;86:854-847. Kasperk CH, Wakley GK, Hierl T, Ziegler R. Gonadal and adrenal androgens are potent regulators of human bone cell metabolism in vitro. J Bone Miner Res. 1997;12:464-471. Benz DJ, Haussler MR, Thomas MA, et al. High affinity androgen binding and androgenic regulation of A I(I)-procollagen and transforming growth factor-beta steady state messenger ribonucleic acid levels in human osteoblast-like osteosarcoma cells. Endocrinology. 1991;128:2723-2730. Hofbauer LC, Khosla S. Androgen effects on bone metabolism: recent progress and controversies. Eur J Endocrinol. 1999; 140:271-286. Kasperk CH, Fitzsimmons R, Strong D, et al. Studies of the mechanisms by which androgens enhance mitogenesis and differentiation in bone cells. J Clin Endocrinol Metab. 1990;71:1322-1329. Bodine PVN, Riggs BL, Spelsberg TC. Regulation of c-fos
Mayo Clin Proc, January 2000, Vol 75 (Supplement)
8. 9. 10.
II. 12.
13. 14.
15.
expression and TGF-b production by gonadal and adrenal andrlli gens in normal osteoblastic cells. J Steroid Biochem Mol Bioi. 1995;52:149-152. . Pivirotto LA, Cissel DS, Keeting PE. Sex hormones mediate interi leukin-Ib production by human osteoblastic HOBIT cells. Mol Cell Endocrinol. 1995;III :67-74. Bellido T, Jilka RL, Boyce BF, et al. Regulation of interleukin-6,. osteoclastogenesis, and bone mass by androgens: the role of the androgen receptor. J Clin Invest. 1995;95:2886-2895. Pilbeam CC, Raisz LG. Effects of androgens on parathyroid hermone and interleukin-I-stimulated prostaglandin production in cultured neonatal mouse calvariae. J Bone Miner Res. 1990; 5: 1183-1188. Harris SA, Enger RJ, Riggs BL, et al. Development and characterization of a conditionally immortalized human fetal osteoblastic cell line. J Bone Miner Res. 1995;10:178-186. Hofbauer LC, Hicok KC, Schroeder MJ, et al. Developmentand characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene. J Cell Biochem. 1997;66:542-551. Wiren KM, Zhang X, Chang C, et al. Transcriptional upregulation of the human androgen receptor by androgen in bone cells. Endocrinology. 1997;138:2291-2300. Hofbauer LC, Ten RM, Khosla S. The anti-androgen, hydroxyflutamide and androgens inhibit interleukin-6 production by an androgen-responsive human osteoblastic cell line. J Bone Miner Res. 1999;14:1330-1337. Gori F, Hofbauer L, Conover CA, Kholsa S. Effects of androgens on the insulin-like growth factor system in an androgen-responsive human osteoblastic cell line. Endocrinology. 1999;140:5579-5586.
The Effects of Androgens on Osteoblast Function In Vitro SUNDEEP KHOSLA,
MD
The relative contributions of androgens and estrogen to bone metabolism are still being defined. To directly assess androgen effects on bone cells, we have developed human osteoblastic cell models expressing the androgen receptor. This approach is distinct from studies that have
used a variety of transformed cell lines, in that the latter studies showed variability in the results. Investigation into androgen effects on bone has been overshadowed by interest in estrogen effects. Mayo Clin Proc. 2000;75(suppl):S51-S54
T
he presence of the estrogen receptor in human bone cells was described more than a decade ago by Eriksen Ind colleagues. I Their work was followed by numerous ~dies on estrogen's effects in vitro on bone cells. A few years later the presence of the androgen receptor ~ bone cells was described by the same group- and was ~ter confirmed by others. Remarkably, this finding was ~t followed by a comparable explosion in studies of ~drogen's effects on bone cells. Perhaps because osteo~rosis is predominantly a women's disease, the study of ~trogen's effects was emphasized instead. Osteoporosis Mso is a disease of men, however, and the effects of andro~ens on bone in either sex have not been studied to the e degree as that of estrogen. Figure 1 depicts the relative levels of estrogen and drogen receptors in human bone cells by sex, expressed molecules per bone cell nucleus. Data from subsequent ~lUdies by others are listed in Table 1. Values are expressed ~or the androgen receptor per cell in primary human ~steoblasts and also for a variety of human osteosarcoma lines. When the effects of androgens on osteoblast prolif~ation were studied, androgens were generally found to [enhance proliferation (Table 2). There are relatively sig~ficant effects on osteoblast proliferation in general and ~so specifically with dihydrotestosterone (DHT) and ldehydroepiandrosterone (DHEA).
therefore, much of this variability could be attributed to methodologic differences. In an effort to explain the variability, it was noted that studies done in primary human osteoblasts, in fact, used bone chips in culture and then examined whatever grew out.' The result-a mixture of fibroblastic cells and osteoblastic cells-demonstrated variable percentages of alkaline phosphatase staining. This raises the question of whether osteoblast-related effects or effects due to other cell types at varying stages of differentiation were being studied. Similar variability arose in systems using rat cells. In cases when osteosarcoma-TE85 cells-had been used, there appeared to be a decrease in proliferation.' So these various systems added controversy te the observations being made about androgen effects on proliferation. If one looks at differentiation markers, namely, alkaline phosphatase or type I collagen, osteocalcin, or mineralization of extracellular matrix, the results have, in general, shown "anabolic" effects of androgen, although these are variable (Table 3). In studies done on primary cultures from bone chips, it was unclear what cell population was being studied. Nonetheless, in general, one sees an increase in the percentage of cells expressing alkaline phosphatase, either an increase or no change in type I collagen and proline incorporation, and either an increase or no change in osteocalcin secretion.> Various growth factors, or cytokines, such as transforming growth factor ~ (TGF-~), also have been studied (Table 4). Androgens, including testosterone, DHT, and DHEA, increased mRNA levels and activity of a number of growth factors.w? The results also showed that the insulin-like growth factor II (IGF-II) receptor was increased by DHT.6 Interleukin 1~ (IL-l~) activity also was increased with androgen stimulation, which is surprising, since, in the reported osteoblastic systems, estrogen has tended to decrease IL-l ~ production.f IL-6 was studied in a preadipocytic marrow cell line, where it appeared that pro-
~
SUMMARY OF ANDROGEN EFFECTS DN BONE CELLS Some of the apparent variability in androgen receptor values probably arises from the methods used to determine the number of binding sites, ie, whether an antibody or a 'specific nuclear binding assay was used. Theoretically, From the Division of Endocrinology, Metabolism, Nutrition, and Internal Medicine, Mayo Clinic Rochester, Rochester, Minn. Presented at Symposium on Testosterone Replacement in Elderly Men, Palm Springs, Calif, March 18-20, 1999. Mayo Clin Proc. 2000;75(suppl):S51-S54
S51
© 2000 Mayo Foundation for Medical Education and Research
The Effects of Androgens on Osteoblast Function In Vitro
S52
4,000
3.000
Moleculesl nucleus 2,000
1,000
Estrogen receptor
a a
••
a
••
~
0
o~o Male
• •
••
•• •• Female
Table 2.-Effects of Androgens on Osteoblast Proliferati«m Cells
•••
a a
a
Androgen receptor
Mayo Clin Proc, January 2000, Vol 75 (Supplement)
0
Ga 000
808 Male
••• ~
•• •
Androgen(s)
Effect
Primary human osteoblasts Primary human osteoblasts
DHT DHT DHEA
t50% t200% t88%
Primary rat diaphyseal osteoblasts Primary rat epiphyseal cells Primary rat calvarial osteoblasts
T T DHT T
tlOO% t57% t 150% t70%
TE-85 osteosarcoma cells
DHT T
.25% .20%
Female
*Adapted from Hofbauer and Khosla.f Figure 1. Relative levels of estrogen and androgen receptors by gender in human bone cells. Adapted from Eriksen et all and Colvard et al.2
duction was decreased by androgens." Prostaglandin Eb another potential mediator of bone resorption with sex steroid deficiency, was decreased by androgens. 10 While the literature contains data pertaining to androgen effects on bone, these effects have not been as well studied as those of estrogen. The data are sparse and are confounded by differences in the model systems that generated them. One can characterize the sources of variability in the data as arising from differences in donor age (fetal, neonatal, adolescent, adult), skeletal site (vertebral, femoral, calvarial), type of bone (cancellous, cortical), species, degree of transformation (osteosarcoma cell lines vs conditionally immortalized cells vs primary cultures), and stage of differentiation of the cells. Variability in species can present important differences, particularly with respect to growth factors. In human bone, the predominant IGF involved in bone metabolism is IGF-II, whereas in rat bone it is IGF-I. Many studies use rat bone. The degree of cellular transformation is another impor-
Table I.-Androgen Receptors in Primary Human Osteoblasts and Human Osteosarcoma Cell Lines* Cells
Characteristics
hOB hOB hOB
Primary human osteoblasts Primary human osteoblasts Primary human osteoblasts
146 ± 19 821 ± 140 5520 ± 240
TE85 U2-0S SaOS-2
Human osteosarcoma cell line Human osteosarcoma cell line Human osteosarcoma cell line
2800-3600 1600-3600 1300-5800
*Adapted from Hofbauer and
Khosla. 5
ARicell
tant variable. Many studies have used osteosarcoma cell lines containing multiple genetic abnormalities. Finally, the stage of differentiation of the osteoblasts has often not been considered. The effects of sex steroids on the mature fully differentiated cell vs the osteoblast precursor cell may be quite different. ' DEVELOPING NEW MODEL SYSTEMS To circumvent some of these problems, several years ago our group developed conditionally immortalized cells derived from a human fetus, known as human fetal osteoblast (hFOB) cells. [1 These cells are immortalized with a temperature-sensitive SV40 large T antigen. At the permissive temperature of 34°C, the T antigen is active, and the cells proliferate; however, because of the temperature-sensitive mutation in the T antigen, the cells decrease proliferating when the ambient temperature reaches 39°C.
Table 3.-Androgen Effects on Osteoblast Differentiation Markers* Marker
Androgen(s)
Alkaline phosphatase (AP) Percentage of AP-positive cells AP activity
DHT DHT,DHEA DHT,T
Type 1 collagen mRNAlevels
DHT,T
(3H)proline incorporation
DHT DHT
Osteocalcin secretion
DHT, T,DHEA DHT, T, DHEA
Mineralization of extracellular matrix
DHT, DHEA
*Adapted from Hofbauer and Khosla.>
Change
.. .. .. .. t
t
t
t t
t
\tayo Clin Proc, January 2000, Vol 75 (Supplement)
The Effects of Androgens on Osteoblast Function In Vitro
Table 4.-Androgen Effects on Growth Factors and
Cytokines*
Androgen(s)
Change
TGF-~
DHT,T DHT, T,DHEA
IGF-IIR
DHT
lL-l~
T
t t t t t
1L-6
DHT, T,DHEA DHT,T
....
PGE2
DHT,T
Cytokine
..
Method mRNAlevels Activity mRNA levels Affinity for IGF-II mRNA levels, protein production Reporter construct mRNA levels, protein production Protein production
*Adapted from Hofbauer and Khosla. 5
They then differentiate and express the complete osteoblastic phenotype, including the formation of mineralized nodules. Even though the primary bone cultures for these cells contained estrogen receptors and androgen receptors, perhaps due to continuous culture in vitro, hFOB cells express low levels of both (200 to 300 receptors per nucleus). This is a limitation, as more robust receptor expression is preferable because it leads to good responses, especially in initial studies. While researchers were searching for a more robust response, another cell line was developed-the hFOBI AR cell line.t? The ideal system comprises primary cultures; however, such cultures are difficult to obtain, they proliferate slowly, and they reflect a great deal of interindividual variability, making data interpretation difficult. They also tend toward senescence with multiple passages, which becomes associated with low levels of sex steroid receptors. By using the "conditionally" immortalized cells grown at the restrictive temperature, we at least have a well-defined clonal population of cells with fully understood characteristics to use for beginning examination of factors regulated by androgens in these cells. The expression plasmid used for developing the hFOBI AR cells used the human androgen receptor (hAR) cloned downstream of the cytomegalovirus promoter.There is an SV40 polyadenylation signal downstream of the hAR. Since FOB cells are already gentamicin resistant due to the introduction of the T antigen and the plasmid that encodes it, a different antibiotic resistance was needed. This plasmid has a hygromycin-resistant gene that allows us to alternate antibiotic selection and maintain both the T antigen and the androgen receptor in continuous culture.
S53
STUDIES WITH THE HFOB/AR CELL LINE AND ITS CLONES In initial studies, the parent hFOB cell line had very low but detectable levels of androgen receptors. Several hFOB/AR clones were not very different, but the clone identified as hFOB/AR-6 had much higher levels, which actually increased when DHT was added. In a chloramphenical acetyl-transferase (CAT), a stimulation of computed tomographic activity was shown going from the absence to the presence of androgen. Some data in the literature suggest that there may be important differences between bone and reproductive tissues in terms of androgen autoregulation of the receptor. In prostate cells, androgen decreases androgen receptor levels, whereas the opposite appears to be the case in bone cells.U It would be quite interesting to further explore these regulatory differences in prostate and bone. Nuclear binding studies revealed that the hFOB/AR cells have approximately 3800 receptors. This has been demonstrated in 2 clones, whereas the parent hFOB cell line expressed about 200 to 300 receptors per cell. This model system, while not ideal, could at least provide clues as to which target genes are being regulated by androgens. We have used hFOB/AR cells to study the effects of androgens on IL-6 production.!! These studies indicate that gonadal but not adrenal androgens markedly reduce basal and cytokine-stirnulated IL-6 production by these cells. We also have examined the regulation of the IOF system in these cells. 15 Thus, gonadal, but not adrenal, androgens increase the production of IOF-I by these cells and also have important effects on the IOF-binding proteins. Further studies with these cells involve using differential display and gene-chip technology in attempting to identify other potential androgen-regulated genes in bone cells. CONCLUSIONS Bone cells are clearly androgen responsive, as seen in both primary bone cell cultures and model systems. Variability in results obtained from in vitro studies is largely due to differences in the model systems used. Within model systems, it is necessary to clearly define and control the sources of such variability (ie, donor and bone type, degree of transformation, and stage of differentiation) and to avoid extrapolation from one species to the other. New model systems, such as the hFOB/AR cells and the comparablehFOBIERcells, are useful for identifying the target genes that are regulatedand for understanding the processes that are involved. However,it is necessary to return to the primary nontransfected systems to verify the results.
S54
The Effects of Androgens on Osteoblast Function In Vitro
ACKNOWLEDGMENTS The work of Francesca Gori, Lawrence Hofbauer, Kevin Hickock, and Cheryl Conover is gratefully acknowledged, as is the contribution of Dr Neary at Schering-Plough.
REFERENCES 1.
2. 3. 4.
5. 6. 7.
Eriksen EF, Colvard DS, Berg NJ, et al. Evidence of estrogen receptors in normal human osteoblast-like cells. Science. 1988; 241:84-86. Colvard DS, Eriksen EF, Keeting PE, et al. Identification of androgen receptors in normal human osteoblast-like cells. Proc Natl Acad Sci USA. 1989;86:854-847. Kasperk CH, Wakley GK, Hierl T, Ziegler R. Gonadal and adrenal androgens are potent regulators of human bone cell metabolism in vitro. J Bone Miner Res. 1997;12:464-471. Benz DJ, Haussler MR, Thomas MA, et al. High affinity androgen binding and androgenic regulation of A I(I)-procollagen and transforming growth factor-beta steady state messenger ribonucleic acid levels in human osteoblast-like osteosarcoma cells. Endocrinology. 1991;128:2723-2730. Hofbauer LC, Khosla S. Androgen effects on bone metabolism: recent progress and controversies. Eur J Endocrinol. 1999; 140:271-286. Kasperk CH, Fitzsimmons R, Strong D, et al. Studies of the mechanisms by which androgens enhance mitogenesis and differentiation in bone cells. J Clin Endocrinol Metab. 1990;71:1322-1329. Bodine PVN, Riggs BL, Spelsberg TC. Regulation of c-fos
Mayo Clin Proc, January 2000, Vol 75 (Supplement)
8. 9. 10.
II. 12.
13. 14.
15.
expression and TGF-b production by gonadal and adrenal andrlli gens in normal osteoblastic cells. J Steroid Biochem Mol Bioi. 1995;52:149-152. . Pivirotto LA, Cissel DS, Keeting PE. Sex hormones mediate interi leukin-Ib production by human osteoblastic HOBIT cells. Mol Cell Endocrinol. 1995;III :67-74. Bellido T, Jilka RL, Boyce BF, et al. Regulation of interleukin-6,. osteoclastogenesis, and bone mass by androgens: the role of the androgen receptor. J Clin Invest. 1995;95:2886-2895. Pilbeam CC, Raisz LG. Effects of androgens on parathyroid hermone and interleukin-I-stimulated prostaglandin production in cultured neonatal mouse calvariae. J Bone Miner Res. 1990; 5: 1183-1188. Harris SA, Enger RJ, Riggs BL, et al. Development and characterization of a conditionally immortalized human fetal osteoblastic cell line. J Bone Miner Res. 1995;10:178-186. Hofbauer LC, Hicok KC, Schroeder MJ, et al. Developmentand characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene. J Cell Biochem. 1997;66:542-551. Wiren KM, Zhang X, Chang C, et al. Transcriptional upregulation of the human androgen receptor by androgen in bone cells. Endocrinology. 1997;138:2291-2300. Hofbauer LC, Ten RM, Khosla S. The anti-androgen, hydroxyflutamide and androgens inhibit interleukin-6 production by an androgen-responsive human osteoblastic cell line. J Bone Miner Res. 1999;14:1330-1337. Gori F, Hofbauer L, Conover CA, Kholsa S. Effects of androgens on the insulin-like growth factor system in an androgen-responsive human osteoblastic cell line. Endocrinology. 1999;140:5579-5586.