This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Issa, S. Articles by Schweikert, H.-U. Search for Related Content PubMed PubMed Citation Articles by Issa, S. Articles by Schweikert, H.-U.

Human Osteoblast-Like Cells Express Predominantly Steroid 5-Reductase Type 1

Department of Internal Medicine, University of Bonn (S.I., D.S., M.F., H.U.S.), Bonn, Germany; Department of Orthopedics, St. Petrus Krankenhaus (L.W.), 53113 Bonn, Germany; Department of Pathology, University of Freiburg (H.E.S.), 79002 Freiburg, Germany; and Department of Molecular Genetics, University of Texas Southwestern Medical Center (D.W.R.), Dallas, Texas 75390-9046

Address all correspondence and requests for reprints to: Hans-Udo Schweikert, M.D., Endocrinology, Department of Internal Medicine, University of Bonn, Wilhelmstrasse 35-37, 53111 Bonn, Germany.

Abstract

In previous studies we established that human bone and human osteoblast-like cells (hOB cells) cultured from bone express 5-reductase (5-R) activity, as demonstrated by the conversion of testosterone and androstenedione to their corresponding 5-reduced metabolites, 5-dihydrotestosterone (DHT) and 5-androstanedione. Two 5-R isozymes (types 1 and 2) have been identified in various tissues. As their nature in bone is unknown, we investigated which isozymes were expressed in first passage hOB cells cultured from bone specimens obtained from six donors (five women and one man). For comparison, 5-reductase isozyme expression in genital skin fibroblasts cultured from foreskin of three males was determined. Pharmacological and biochemical studies using selective inhibitors of the 5-R isozymes were performed, and gene expression was assessed by RT-PCR. In hOB cells, LY191704, a potent nonsteroidal selective inhibitor of 5-R type 1, and the 4-azasteroid 17ß-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5-androstan-3-one (a dual inhibitor of 5-R types 1 and 2) inhibited 5-R activity with a 50% inhibitory concentration (IC₅₀) of approximately 4 nM. Finasteride, a selective inhibitor of 5-R type 2, blocked 5-R activity with an IC₅₀ of approximately 60 nM. The IC₅₀ of progesterone, a physiological substrate for 5-R, was approximately 200 nM. In genital skin fibroblasts, LY191704 inhibited 5-R with an IC₅₀ of more than 5000 nM, whereas finasteride and 17ß-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5-androstan-3-one effectively inhibited 5-R with IC₅₀ of approximately 4 nM. Experiments to determine 5-reductase activity in homogenates of hOB cells as a function of pH showed very low activity at pH 5.5, but a broad shoulder of activity from pH 6.0–9.0, which was not inhibited by finasteride, but was nearly completely blocked by LY191704. RT-PCR revealed that 5-R type 1 and 2 mRNAs were expressed in both bone and genital skin fibroblasts. Based on our pharmacological and biochemical studies, it appears that 5-R activity in hOB cells is catalyzed predominantly by the type 1 rather than the type 2 isozyme. This expression pattern is in contrast to that in genital skin fibroblasts, where the activity of the type 2 isozyme prevails. As in most androgen target tissues DHT is biologically more active as an androgen than testosterone, DHT is formed in bone by 5-R type 1 action from circulating testosterone, and bone cells also express the androgen receptor, local DHT production may play a physiological role in human bone homeostasis.

ANDROGENS AFFECT SKELETAL maturation and exert profound effects on the homeostasis of mature bone. In men, androgen deficiency is associated with premature bone loss (1) and an increased frequency of osteoporotic fractures (2). Testosterone substitution increases or stabilizes bone mass in hypogonadal men (3). In postmenopausal women treatment with anabolic steroids increases bone mass (4, 5), and circulating concentrations of androgens have been reported to be predictors of bone mineral density in women (6). Furthermore, androgens have been shown to stimulate the proliferation of cultured murine and human osteoblast-like cells (hOB cells) (7), and androgen receptors have been identified in cultured hOB cells (8, 9, 10). Although androgens at the molecular level act similarly to other steroid hormones, i.e. by combining with a specific receptor, their mechanism of action is complicated by the fact that in most androgen target tissues testosterone is converted by the enzyme steroid 5-reductase (5-R) to 5-dihydrotestosterone (DHT) before binding to the androgen receptor. Thus, any study to unravel androgen action must take into account the metabolism of the hormone within the target tissue or cell type studied. Previously we showed that both ground fragments of human bone and cultured human osteoblast-like cells (hOB cells) express 5-R activity and thus can form DHT from circulating androgen precursors (11, 12). Two 5-R isozymes, designated types 1 and 2, have been identified. In addition to structural differences, the two enzymes differ with respect to their biochemical properties, pharmacologies, genetics, and tissue distribution (13, 14). As the nature of the isozymes expressed in bone remains unknown, we used pharmacological inhibitors and RT-PCR to assess 5-R isozyme expression patterns in cultured hOB cells. The data show that the 5-R type 1 isozyme predominates in this cell type.

Materials and Methods

Materials

Silica gel thin layer chromatography sheets with a plastic back (Polygram Sil G-Hy) were obtained from Machery and Nagel (Duren, Germany). [1,2,6,7-³H]Androstenedione (87.0 Ci/mmol) and [1,2,6,7-³H]testosterone (98.0 Ci/mmol) were purchased from Perkin-Elmer NEN Life Science Products (Brussels, Belgium). Tritium-labeled steroids were purified by thin layer chromatography using the solvent system dichloromethane/ethyl acetate/methanol (85:15:2, vol/vol/vol) to assure they were more than 95% pure. Nonradioactive steroids (5-androstanedione, androstenedione, 5-androstane-3,17ß-diol, 5-androstane-3ß,17ß-diol, DHT, testosterone, and progesterone) were purchased from Merck \|[amp ]\| Co., Inc. (Darmstadt, Germany). The 5-R inhibitors LY191704 (8-chloro-4-methyl-1,2,3,4,4,5,6,10ß-octaahydro-benzol[f]quinolin-3(2H)-one), 17ß-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5-andro-stan-3-one (4-MA), and finasteride [17ß-(N-tert-butylcarbamoyl)-4-aza-5-androst-1-en-3-one) were gifts from Eli Lilly \|[amp ]\| Co. (Indianapolis, IN; LY191704) and Merck Research Laboratories (Rahway, NJ; 4-MA and finasteride).

MEM, penicillin, streptomycin, and nonessential amino acids were obtained from Life Technologies, Inc. (Karlsruhe, Germany). Dulbecco’s PBS was purchased from Seromed (Berlin, Germany). Fetal calf serum was obtained from Cytogen (Ober-Morlen, Germany). Tissue culture dishes (Falcon Primaria; 60 x 15 mm; polystyrole with a modified surface) were obtained from BD Biosciences (Heidelberg, Germany). Taq polymerase and ribonuclease-free deoxyribonuclease I were purchased from Roche (Mannheim, Germany). TRIzol reagent and the Superscript preamplification system for first-strand cDNA synthesis, Superscript II RT, and 7-deaza-dGTP were obtained from Life Technologies, Inc. Primers were obtained from MWG Biotech (Ebersberg, Germany). All other chemicals were reagent grade or better and were used as supplied by the manufacturer.

Source of tissue and cell culture

Bone cells. Cell strains were cultured from bone removed at orthopedic surgery from five postmenopausal women (age range, 55–95 yr) and a 58-yr-old man. Femoral tissue was obtained from bone discarded during hip or knee replacement for osteoarthritis. The bone was used with the informed written consent of the patients. Ethical approval was obtained from the ethics committee of the medical faculty of University of Bonn (Bonn, Germany). Bone fragments free from bone marrow were prepared for bone cell culture as previously described (11, 15). In brief, after removal the bone was placed in ice-cold saline and taken to the laboratory for immediate processing. A biopsy of every bone sample was taken for later histological analysis. Inflammation, malignancy, and/or necrosis of the bone were excluded. The cancellous bone was removed from the open end of the femur with a curette, dissected into fine pieces, and ground with a mortar. Throughout, the tissue was thoroughly cleaned of bone marrow by rinsing with chilled and then 20 C warmed isotonic saline until fine white, ivory-colored bone fragments remained.

Genital skin fibroblasts. The fibroblast strains used in this study were derived from explants of foreskin from three males (aged, 1, 5, and 23 yr) undergoing circumcision or repair of developmental defects of the urogenital tract. Biopsies of tissue from minors were performed with the consent of the parents.

Cell culture and phenotypic characteristics of bone cells

The methods used for bone cell culture were reported previously (12). In all experiments hOB cells were used in the first passage. Skin fibroblast strains were established and cultured as previously described (16, 17, 18). Cells were used between passages 6 and 8.

The cultured bone cells displayed typical osteoblastic features, such as alkaline phosphatase expression, secretion of osteocalcin after stimulation with 1,25-dihydroxycholecalciferol, cAMP secretion that increased after PTH treatment, and mineralization of extracellular bone matrix (12). In contrast, alkaline phosphatase activity was 12-fold lower in skin fibroblasts cultured from either genital or nongenital skin, and the cells did not secrete osteocalcin upon exposure to 1,25-dihydroxycholecalciferol.

Determination of 5-R activity

Whole cell assay. hOB cells were incubated for 6 h and genital skin fibroblasts for 3 h with 500 nM androstenedione (50 nM ³H-labeled and 450 nM unlabeled steroid) in the presence or absence of increasing concentrations of LY191704, 4-MA, finasteride, or progesterone (0.5–5000 nM). After incubation, the medium was extracted with chloroform/methanol (2:1, vol/vol), and the steroids were separated by thin layer chromatography (12). The DNA content of the cells was determined as previously described (12). 5-R activity (sum of 5-androstanedione, DHT, and 5-androstanediols) was determined. Enzyme activities in the presence of inhibitors were expressed as a percentage of 5-R activities in the absence of these agents. In all instances 5-R activity was determined in duplicate samples.

Cell free extract assay. Determination of pH optima of 5-R activity in cell-free extracts of hOB cells was tested as previously described (19) using the following modifications. The incubation mixture contained [1,2,6,7-³H]androstenedione (100 nM), NADPH (3 mM), citrate buffer (pH 4.5–9.0), and 125 µg protein in a total volume of 0.2 ml. Incubations were conducted in duplicate for 1 h in the presence or absence of either LY191704 (100 nM) or finasteride (100 nM) in a shaking water bath at 37 C. The reaction was terminated by the addition of 5 vol chloroform/methanol (2:1, vol/vol), and steroids were extracted and separated as previously described (12).

mRNA assessment

Total RNA was isolated from cells using TRIzol reagent according to the manufacturer’s instructions. Residual genomic DNA was removed from RNA preparations by incubating the samples for 1 h at 37 C in buffer (pH 7.2) made with 25 mM Tris-HCl, 5 mM MgCl₂, 0.1 mM EDTA, and 2 U ribonuclease-free deoxyribonuclease I/µg RNA. This incubation was followed by a second purification using TRIzol reagent.

cDNA synthesis was performed using the Superscript preamplification system for first-strand cDNA synthesis. A 20-µl reaction mixture contained 20 mM Tris-HCl (pH 8.4); 50 mM KCl; 2.5 mM MgCl₂; 10 mM dithiothreitol; 0.5 mM each of deoxy (d)-ATP, dCTP, dGTP, and dTTP; 0.5 µg oligo(deoxythymidine)_12–18 primer; 1 µg total RNA; and 200 U Superscript II RT. Mixtures were incubated at 42 C for 55 min and then stored at -20 C.

PCRs were performed using a GeneAmp PCR System 2400 (PE Applied Biosystems, Langen, Germany). A 20-µl reaction mixture contained 10 mM Tris-HCl (pH 8.3); 1.5 mM MgCl₂; 50 mM KCl; 0.25 mM each of dATP, dCTP, dGTP, and dTTP; 0.3 µM of the corresponding specific sense and antisense primers (Table 1); 2 µl cDNA; and 0.5 U Taq DNA polymerase. For amplification of 5-R type 2-specific cDNA, dGTP containing 2% 7-deaza-dGTP was used.

Nested PCR was performed as described above, except using the corresponding nested primers (0.3 µM each) and 2 µl of a 1:5 dilution of the product of the first PCR as template. Cycling conditions were the same as described above.

Aliquots of the samples were subjected to agarose gel electrophoresis. The identity of the reaction product was verified by sequence analysis (MWG Biotech).

Results

To determine 5-R isozyme expression in human bone, we analyzed enzyme activity and gene expression in hOB cells cultured from bone of six donors (five women and one man) and, for comparison, in cultured genital skin fibroblast strains derived from three males. The findings of pharmacological studies using selective and nonselective inhibitors of the 5-R isozymes are presented in Fig. 1 and Table 2. Both LY191704 (a selective 5-R type 1 inhibitor) and 4-MA (a dual inhibitor of 5-R type 1 and type 2) blocked enzyme activity in bone cells with an IC₅₀ of approximately 4 nM. In contrast, finasteride (a selective 5-R type 2 inhibitor) blocked 5-R activity with an IC₅₀ value of approximately 60 nM. The IC₅₀ value of progesterone, a physiological substrate for 5-R, was about 200 nM. The efficacies of LY191704, 4-MA, and finasteride to inhibit 5-R activity in genital skin fibroblasts are summarized in Table 2 and illustrated in Fig. 2. Finasteride and 4-MA were the most effective inhibitors, with IC₅₀ values of approximately 4 nM. In contrast, LY191704 inhibited 5-R activity with an IC₅₀ of more than 5000 nM. Experiments to determine 5-reductase activity in homogenates as a function of pH (Fig. 3) showed a very low activity at pH 5.5, but a broad shoulder of activity from pH 6.0–9.0, which was not inhibited by finasteride, but was nearly completely blocked by LY191704.

View larger version (21K): [in this window] [in a new window]   Figure 1. Inhibition of 5-R activity in human osteoblast-like cells by LY191704, 4-MA, finasteride, and progesterone. 5-R activity was measured in six cell strains derived from bone from six patients undergoing orthopedic surgery. Cells from each strain were exposed to 500 nM androstenedione in the presence or absence of increasing concentrations of LY191704, 4-MA, finasteride (0.5–5000 nM), and progesterone (5–5000 nM), respectively. After incubation for 6 h, the medium was extracted into organic solvent, and the samples were analyzed by thin layer chromatography as described. In each instance 5-R activity was determined in duplicate samples. The relative 5-R activity (compared with control levels) at each concentration is illustrated as the mean ± SEM.

View this table: [in this window] [in a new window]   Table 2. IC50 values as obtained with various 5-R inhibitors and selectivity in both osteoblast-like cells (hOB cells) and genital skin fibroblasts (GSF)

View larger version (21K): [in this window] [in a new window]   Figure 2. Inhibition of 5-R activity in genital skin fibroblasts from explants of foreskin obtained from three males at 1, 5, and 23 yr of age undergoing circumcision or repair of hypospadias and phimosis, respectively. Cells from each strain were exposed to 500 nM androstenedione in the presence or absence of increasing concentrations of LY191704, 4-MA, finasteride, and progesterone (0.5–5000 nM), respectively. Incubation of cells and determination of the relative 5-R activity were performed as described in Fig. 1.

View larger version (18K): [in this window] [in a new window]   Figure 3. 5-R activity in human osteoblast-like cells as a function of pH. Cells were grown and homogenized, and 5-R activity was assayed in the presence or absence of either finasteride or LY191704 as described in the text.

View larger version (68K): [in this window] [in a new window]   Figure 4. Gene expression of 5-R types 1 and 2 in hOB cells and genital skin fibroblasts. A, 5-R type1; B, 5-R type 2. M, 100-bp ladder; 1–3, three different hOB cell strains (A) and GSF cell strains (B), respectively; 4, negative control.

Based on pharmacological, biochemical, and molecular-biological studies we demonstrated that hOB cells cultured from bone of both men and women contain predominantly the 5-R isozyme type 1. This finding further substantiates and expands our previous studies aimed at understanding the basic mechanisms by which circulating steroids exert their osteoprotective actions in mature human bone. In those studies we demonstrated the presence of the enzymes 5-R, 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and aromatase in both ground fragments of human bone and cultured hOB cells. We found that androstenedione and testosterone were aromatized to form estrogens and also served as substrates for the 5-R to undergo irreversible reduction to 5-androstanes, the principal one of which is DHT (11, 12, 15, 20). DHT is formed in bone from testosterone by 5-R action as well as from the androgen precursor androstenedione by 5-R and 17ß-HSD action (11, 12). Recently we have further characterized 17ß-HSD in hOB cells and identified mRNA expression of the 17ß-HSD isozymes types 1–4 (21). In addition, we assessed in both bone fragments and hOB cells the conversion of estrone sulfate to estrone and estradiol, thus demonstrating the consecutive action of the enzymes steroid sulfatase and 17ß-HSD (22). Thus, bone cells express all of the enzymes required to convert circulating precursors to biological potent estrogens, such as estradiol, and to androgens, such as testosterone and DHT.

In this study we have further characterized the 5-R isozymes present in bone cells. To this end 5-R isozymes were first differentiated on the basis of their sensitivity to inhibitors. The inhibitory effects of LY191704, a highly selective 5-R isozyme type 1 inhibitor; 4-MA, a dual type 1 and 2 inhibitor; and finasteride, a selective type 2 inhibitor (23, 24), were determined in hOB cells and, for comparison, in genital skin fibroblasts, which are known to express predominantly the type 2 isozyme. Assessing the corresponding IC₅₀ values of these compounds, we found that LY191704 had a selectivity ratio (IC₅₀ 5-R type 1 vs. IC₅₀ 5-R type 2) for inhibiting 5-R in hOB cells vs. genital skin fibroblasts exceeding 1250; the corresponding values for 4-MA and finasteride were approximately 0.75 and 0.07, respectively. These data are consistent with the predominant presence of the 5-R type 1 isozyme in hOB cells and are in agreement with reported potencies against prostate, scalp, and cloned 5-R type 1 isozyme (23, 24). Previous studies have shown that the two 5-R isozymes have different pH optima. The type 1 isozyme has a broad pH optimum (pH 6.0–9.0), whereas the type 2 isozyme has a narrow acidic pH optimum centered around 5.0 (13, 14). The absence of enzymatic activity at pH 4.5, the very low enzymatic activity at pH 5.5, and the broad pH optimum pattern from 6–9, which was nearly completely blocked by LY191704, but remained unaffected by finasteride, clearly substantiates that the type 1 is the predominantly active enzyme in hOB cells.

Furthermore, the presence of the 5-R type 1 isozyme in hOB cells was demonstrated by molecular biological studies. mRNAs encoding both 5-R type 1 and 2 were detected in all bone cell strains examined. This result might reflect the relative sensitivities of the RT-PCR compared with the biochemical assay and/or it might be due to the fact that mRNA is modulated at a later step of its intracellular signal pathway. Our results thus show that 5-R type 1 is predominantly present in hOB cells. 5-R type 1 as the predominant 5-R isozyme has been identified previously in a variety of other human tissues, such as skin, kidney, and brain (25, 26). In skin it is transiently expressed in newborn skin and scalp; it is permanently expressed in skin after the onset of puberty (25). In tissues such as the prostate, seminal vesicle epididymis, and medulla oblongata, type 2 appears to be the predominant 5-R isozyme. Interestingly, neither isozyme expression could be detected in skeletal muscle (25), although a major fraction of androgen in human skeletal muscle is DHT, which implies that it is derived from the circulation (27, 28). Furthermore, DHT stimulated muscle growth in castrated animals (29). It yielded limited improvement in lower limb muscle strength in a double blind, placebo-controlled clinical trial in older men with partial androgen deficiency. The men were treated during 3 months with a transdermally applied DHT gel (30).

Our studies further show that the 5-R type 2 inhibitor finasteride is a poor inhibitor of 5-R activity in hOB cells. These findings are in agreement with studies in rodents (31) and humans (32, 33) designed to address the impact of finasteride on skeletal integrity. These studies showed that finasteride did not have significant effects on parameters of bone remodeling and bone mineral densities (BMD). In interpreting these results, however, it should be considered that the prospective value of both currently available clinical studies is limited due to their relatively small patient size. Furthermore, as finasteride is taken permanently to exert therapeutic effects, the time of observation in these studies (1–2 yr) was short. An alternative explanation of why finasteride might not exert unfavorable side-effects on bone would be that even if the drug in bone cells should inhibit DHT formation, this block could render higher levels of intracellular aromatizable androgens for estrogen formation. An increase in estrogen could compensate for impaired DHT formation. Further studies are needed to test this hypothesis.

Recently, the impact of androgens on bone has become a matter of debate, as decreased bone density has been observed in two men with mutations of the aromatase gene (34) and also in a man with insensitivity to estrogens due to a single point mutation of the estradiol receptor gene that resulted in a premature stop codon (35). Our demonstration of 5-R activity in bone fragments and cultured bone cells (11, 12) and the current identification of the 5-R type 1 isozyme in human bone cells provide supporting evidence that DHT is, in addition to estrogen, a direct mediator of sex steroid action within human bone. This view is based on the following observations.

First, in vitro studies on direct effects of androgens on bone cells derived from varies sources, such as malignant and normal bone obtained from fetal or mature skeleton in animals and humans, have been reported and have recently been reviewed (36). With regard to our study, the effects of DHT observed in mature hOB cells are of special interest. Kasperk et al. (7) have shown an increase in osteoblast proliferation after treatment with DHT. Growth stimulation was observed in osteoblastic cells derived from mouse calvaria, a human osteosarcoma cell line, and primary hOB cells. Growth was inhibited by the administration of steroidal and nonsteroidal antiandrogens. Further studies have extended these results. DHT pretreatment of hOB cells with DHT enhanced the mitogenic response of fibroblast growth factor and IGF-II. It also caused an increase in IGF-II binding to hOB cells (37).

In vivo studies also revealed direct effects of androgens on bone. For example, Vanderschueren et al. (38) have demonstrated that aromatizable and nonaromatizable androgens prevented bone loss in aged male rats. Recently, Falahati-Nini et al. (39) have assessed the relative contributions of testosterone and estrogen to the regulation of bone resorption in elderly men. The researchers demonstrated that estradiol was the dominant sex steroid regulating bone resorption, whereas both estrogen and testosterone were important in maintaining bone formation (39).

Second, specific high affinity androgen receptors have been identified in hOB cells using methyltrienolone (8) or DHT (9) as ligands as well as in human bone using specific monoclonal antibodies to the human androgen receptor (10).

Third, DHT has been found to be biologically more potent than testosterone, because DHT binds more tightly to the androgen receptor, and the DHT-receptor complex is more readily transferred to the DNA-binding state and activates a receptor reporter gene more efficiently than testosterone (reviewed in Refs. 40 and41). In addition, DHT may regulate specific genes that do not respond to testosterone (42). DHT has been found to up-regulate androgen receptor steady state mRNA level expression and androgen receptor promoter activity in human osteoblastic osteosarcoma cells (43). Consequently, DHT amplifies a weak androgen signal and may also regulate specific genes.

Fourth, as in most, if not all, androgen target organs, DHT is the most active intracellular androgen when 5-R activity is present, our results strongly indicate that DHT is a direct mediator of androgen action within bone.

This assumption is further substantiated by studies assessing BMD in subjects with male pseudohermaphroditism due to insensitivity to androgens and in individuals with 5-R type 2 deficiency. Both disorders are natural models to assess the mechanism of action in its target organs, one of which is bone. Individuals with complete androgen insensitivity syndrome (AIS) are characterized by a 46,XY karyotype; testes that are located intraabdominally, inguinally, or in the labia majora; and an unambiguously female phenotype. In the adult, gonadotropin secretion and testosterone production are in the upper range for men or elevated, and estrogen production is increased (44). The underlying defects of the disorder are mutations in the androgen receptor gene. In several studies BMDs in individuals with this syndrome have been found to be significantly lower than those in age-matched male controls (45, 46, 47). In a recent study BMDs were superior in six subjects with partial AIS compared with those in 22 patients with complete AIS (47). In addition, we have observed in a man with partial AIS due to a mutation in the DNA-binding domain of the androgen receptor that his BMD increased with high dose testosterone therapy, whereas serum estradiol levels remained unaffected (48). Taken together, these data imply that estrogens alone do not fully compensate for the loss of androgen receptor function and support the assumption that androgens exert a direct effect on bone.

In 5-R deficiency, 46,XY gonadal males exhibit ambiguous genitalia caused by mutations in the 5-R type 2 gene. In the adult, serum gonadotropin levels are within the normal range or slightly elevated, and testosterone and estrogen production are similar to those in normal men. Serum DHT levels are either subnormal or may be in the low normal range. Studies of the origin of circulating DHT in these patients showed that the hormone must be contributed predominantly by the 5-R type 1 (40).

BMD was determined in 16 individuals with 5-R type 2 deficiency and in 12 patients with the complete AIS phenotype by Schwartz et al. (49). In men with 5-R type 2 deficiency mean BMD was found to be slightly, but not significantly, decreased at the spine and was not different at the femoral neck compared with normal male controls. In contrast, in subjects with complete AIS, BMD was substantially decreased at the spine and moderately at the hip compared with the corresponding values in age-matched normal males. Our finding that type 1, not type 2, is the predominantly active form of the 5-R enzyme in hOB cells might explain the near-normal BMD observed in men with 5-R deficiency, which is compatible with the possibility that androgen action in bone might be mediated at least to some extent by DHT formed by 5-R type 1 expression.

In summary, we have demonstrated that 5-R type 1 is the predominantly active enzyme in human osteoblast-like cells. As in most androgen target tissues DHT is biologically more active as an androgen than testosterone, DHT is formed in bone from circulating testosterone by 5-R action as well as from the androgen precursor androstenedione by 5-R and 17-HSD action, and bone cells also express the androgen receptor, it appears reasonable to conclude that local DHT production plays a physiological role in human bone homeostasis.

Acknowledgments

We thank Dr. Gabriela Romalo for performing the pH optima experiments, and Dr. Leon Milewich (Dallas, TX) for critical reading of the manuscript. Mrs. Margarete Sudmann provided able technical assistance in the performance of these studies.

Footnotes

This work was supported by grants from the Deutsche Forschungsgemeinschaft (Schw 168/7-2), Bundesministerium für Bildung und Forschung, and NIH (DK-47657). Presented in part at the 81st Annual Meeting of The Endocrine Society, 1999.

Abbreviations: AIS, Androgen insensitivity syndrome; BMD, bone mineral density; d, deoxy-; DHT, 5-dihydrotestosterone; hOB cells, human osteoblast-like cells; 17ß-HSD, 17ß-hydroxysteroid dehydrogenase; IC₅₀, 50% inhibitory concentration; 4-MA, 17ß-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5-androstan-3-one; 5-R, 5-reductase.

Received November 28, 2001.

Accepted July 16, 2002.

References

1. Stepan JJ, Lachman M, Zverina J, Pacovsky V, Baylink DJ 1989 Castrated men exhibit bone loss: effect of calcitonin treatment on biochemical indices of bone remodeling. J Clin Endocrinol Metab 69:523–527[Abstract/Free Full Text]
2. Finkelstein JS, Klibanski A, Neer RM, Greenspan SL, Rosenthal DI, Crowly WF 1987 Osteoporosis in men with idiopathic hypogonadotropic hypogonadism. Ann Intern Med 106:354–361
3. Behre HM, Kliesch S, Leifke E, Link TM, Nieschlag E 1997 Long-term effect of testosterone on bone mineral density in hypogonadal men. J Clin Endocrinol Metab 82:2386–2390[Abstract/Free Full Text]
4. Chesnut CH III, Ivey JL, Gruber HE, Mathews M, Nelp WB, Sisom K, Baylink DJ 1983 Stanozolol in postmenopausal osteoporosis: therapeutic efficacy and possible mechanism of action. Metabolism 32:571–580[CrossRef][Medline]
5. Gennari C, AgnusDei D, Gonelli S, Nardi P 1989 Effects of nandrolone decanoate therapy on bone mass and calcium metabolism in women with established post-menopausal osteoporosis: a double-blind placebo-controlled study. Maturitas 11:187–197[CrossRef][Medline]
6. Buchanan JR, Hospodar P Myers C, Leuenberger P, Demers LM 1988 Effect of excess endogenous androgens on bone density in young women. J Clin Endocrinol Metab 67:937–943[Abstract/Free Full Text]
7. Kasperk CH, Wergedal JE, Farley JR, Linkhart TA, Turner RT, Baylink DJ 1989 Androgens directly stimulate proliferation of bone cells in vitro. Endocrinology 124:1576–1578[Abstract/Free Full Text]
8. Colvard DS, Eriksen EF, Keeting PE, Wilson EM, Lubahn DB, French FS, Riggs BL, Spelsberg TC 1989 Identification of androgen receptors in normal human osteoblast-like cells. Proc Natl Acad Sci USA 86:854–857[Abstract/Free Full Text]
9. Liesegang P, Romalo G, Sudmann M, Wolf L, Schweikert HU 1994 Human osteoblast-like cells contain specific, saturable, high affinity glucocorticoid-, androgen-, estrogen-, and 1,25-dihydroxycholecalciferol receptors. J Androl 15:194–99[Abstract/Free Full Text]
10. Abu EO, Horner A, Kusec V, Triffitt JT, Compston JE 1997 The localization of androgen receptors in human bone. J Clin Endocrinol Metab 82:3493–3497[Abstract/Free Full Text]
11. Schweikert HU, Rulf W, Niederle N, Schaefer HE, Keck E, Krück F 1980 Testosterone metabolism in human bone. Acta Endocrinol (Copenh) 95:258–264
12. Bruch HR, Wolf L, Budde R, Romalo G, Schweikert HU 1992 Androstenedione metabolism in cultured human osteoblast-like cells. J Clin Endocrinol Metab 75:101–105[Abstract]
13. Jenkins EP, Andersson S, Imperato-McGinley J, Wilson JD, Russell DW 1992 Genetic and pharmacological evidence for more than one human steroid 5-reductase. J Clin Invest 89:293–300
14. Russell DW, Wilson JD 1994 Steroid 5-reductase: two genes/two enzymes. Annu Rev Biochem 63:25–61[Medline]
15. Schweikert HU, Wolf L, Romalo G 1995 Aromatization of androstenedione in human bone. Clin Endocrinol (Oxf) 43:37–42[Medline]
16. Schweikert HU, Milewich L, Wilson JD 1976 Aromatization of androstenedione by cultured human fibroblasts. J Clin Endocrinol Metab 43:785–795[Abstract/Free Full Text]
17. Schweikert HU, Schlüter M, Romalo G 1989 Intracellular and nuclear binding of [³H]dihydrotestosterone in cultured genital skin fibroblasts. J Clin Invest 83:662–668
18. Staib P, Kau N, Romalo G, Schweikert HU 1994 Oestrogen formation in genital and non-genital skin fibroblasts cultured from patients with hypospadias. Clin Endocrinol (Oxf) 41:237–243[Medline]
19. Leshin M, Griffin JE, Wilson JD 1978 Hereditary male pseudohermaphroditism associated with an unstable form of 5-reductase. J Clin Invest 62:685–691
20. Romalo G, Sudmann M, Wolf L, Helpenstein-Michels G, Schweikert HU, Aromatization of androstenedione in human osteoblast-like cells. [Abstract 1104]. Proc of the 75th Annual Meeting of The Endocrine Society, Las Vegas, NV, 1993.
21. Feix M, Wolf L, Schweikert HU 2001 Distribution of 17ß-hydroxysteroid dehydrogenases in human osteoblast-like cells. Mol Cell Endocrinol 171:163–164[CrossRef][Medline]
22. Muir M, Romalo G, Sudmann M, Feix M, Wolf L, Schweikert HU, Estrogen formation from estrone sulfate in human bone [Abstract P1244]. Proc of the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000.
23. Hirsch KS, Jones CD, Audia JE, Andersson S, McQuaid, L, Stamm NB, Neubauer BL, Pennigton P, Toomey RE, Russell DW 1993 LY191704: a selective, nonsteroidal inhibitor of human steroid 5-reductase type 1. Proc Natl Acad Sci USA 90:5277–5281[Abstract/Free Full Text]
24. Neubauer BL, Gray HM, Hanke CW, Hirsch KS, Hsiao KC, Jones CD, Kumar MV, Lawhorn DE, Lindzey J, McQuaid L, Tindall DJ, Toomey RE, Yao, C, Audia JE 1996 LY191704 inhibits type I steroid 5-reductase in human scalp. J Clin Endocrinol Metab 81:2055–2060[Abstract]
25. Thigpen AE, Silver RI, Guileyardo JM, Casey ML, McConnell JD, Russell DW 1992 Tissue distribution of steroid 5-reductase isozyme expression. J Clin Invest 92:903–910[CrossRef]
26. Stoffel-Wagner B, Watzka M, Steckelbroeck S, Wickert L, Schramm J, Romalo G, Klingmüller D, Schweikert HU 1998 Expression of 5-reductase in the human temporal lobe of children and adults. J Clin Endocrinol Metab 83:3636–3642[Abstract/Free Full Text]
27. Deslypere JP, Sayed A, Verdonck L, Vermeulen A 1980 Androgen concentration in sexual and non-sexual skin as well as in striated muscle in man. J Steroid Biochem 13:1455–1458[CrossRef][Medline]
28. Wilson JD 1996 Role of dihydrotestosterone in androgen action. Prostate 6(Suppl):88–92
29. Wilson JD 1972 Recent studies on the mechanism of action of testosterone. N Engl J Med 287:1284–1291
30. Ly LP, Jimenez M, Zhuang TN, Celermajer DS, Conway AJ, Handelsman DJ 2001 A double-blind, placebo-controlled, randomized clinical trial of transdermal gel on muscular strength, mobility, and quality of life in older men with partial androgen deficiency. J Clin Endocrinol Metab 86:4078–4088[Abstract/Free Full Text]
31. Rosen HN, Tollin S, Balena R, Middlebrooks VL, Moses AC, Yamamoto M, Zeind AJ, Greenspan SL 1995 Bone density is normal in male rats treated with finasteride. Endocrinology 1236:1381–1387
32. Matzkin H, Chen J, Weisman Y, Goldray D, Pappas F, Jaccard N, Brav Z 1992 Prolonged treatment with finasteride (a 5-reductase inhibitor) does not affect bone density and metabolism. Clin Endorinol (Oxf) 37:432–436[CrossRef]
33. Tollin SR, Rosen HN, Zurowski K, Saltzman B, Zeind AJ, Berg S, Greenspan SL 1996 Finasteride does not alter bone turnover in men with benign prostatic hyperplasia–a clinical research center study. J Clin Endocrinol Metab 81:1031–1034[Abstract]
34. Morishima A, Grumbach MM, Simpson ER, Fisher C, Quin K 1995 Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. J Clin Endocrinol Metab 80:3689–3698[Abstract]
35. Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB, Korach KS 1994 Estrogen resistance caused by a mutation in the estrogen receptor gene in a man. N Engl J Med 331:1056–1061 (Erratum: N Engl J Med. 1995, 332:131)[Abstract/Free Full Text]
36. Hofbauer L, Khosla S 1999 Androgen effects on bone metabolism: recent progress and controversies. Eur J Endocrinol 140:271–286[Abstract]
37. Kasperk C, Fitzsimmons R, Strong D, Mohan S, Jennings J, Wergedal, J, Baylink D 1990 Studies of the mechanism by which androgens enhance mitogenesis and differentiation in bone cells. J Clin Endocrinol Metab 71:1322–1329[Abstract/Free Full Text]
38. Vanderschueren D, Herck EV, Suiker AMH, Visser WJ, Schot LPC, Bouillon R 1992 Bone and mineral metabolism in aged male rats: short and long term effects of androgen deficiency. Endocrinology 130:2906–2916[Abstract/Free Full Text]
39. Falahati-Nini A, Riggs BL, Atkinson EJ, O’Fallon WM, Eastell R, Khosla S 2000 Relative contribution of testosterone and estrogen in regulating bone resorption in normal elderly men. J Clin Invest 106:1553–1560[Medline]
40. Wilson JD, Griffin JE, Russell DW 1993 Steroid 5-reductase 2 deficiency. Endocr Rev 14:577–593[Abstract/Free Full Text]
41. McPhaul MJ, Young M 2001 Complexities of androgen action. J Am Acad Dermatol 45:S87–S94
42. George FW, Russell DW, Wilson JD 1991 Feed-forward control of prostate growth: dihydrotestosterone induces expression of its own biosynthetic enzyme, steroid 5-reductase. Proc Natl Acad Sci USA 88:8044–8047[Abstract/Free Full Text]
43. Wiren KM, Zhang X, Chang C, Keenan E, Orwoll ES 1997 Transcriptional up-regulation of the human androgen receptor by androgen in bone cells. Endocrinology 138:2291–2300[Abstract/Free Full Text]
44. MacDonald PC, Madden JD, Brenner PF, Wilson JD, Siiteri PK 1979 Origin of estrogen in normal men and in women with testicular feminization. J Clin Endocrinol Metab 49:905–916[Abstract/Free Full Text]
45. Bertelloni S, Baroncelli GI, Federico G, Cappa M, Lala R, Saggese G 1998 Altered bone mineral density in patients with complete androgen insensitivity syndrome. Horm Res 50:309–314[CrossRef][Medline]
46. Mizunuma H, Soda M, Okano H, Kagami I, Miyamoto S, Ohsawa M, Ibuki Y 1998 Changes in bone mineral densities after orchidectomy and hormone replacement therapy in individuals with androgen insensitivity syndrome. Hum Reprod 13:2816–2818[Abstract/Free Full Text]
47. Marcus R, Leary D, Schneider DL, Shane E, Favus M, Quigley CA 2000 The contribution of testosterone to skeletal development and maintenance: lessons from the androgen insensitivity syndrome. J Clin Endocrinol Metab 85:1032–1037[Abstract/Free Full Text]
48. Weidemann W, Peters B, Romalo G, Spindler KD, Schweikert HU 1998 Response to androgen treatment in a patient with partial androgen insensitivity and a mutation in the deoxyribonucleic acid-binding domain of the androgen receptor. J Clin Endocrinol Metab 83:1173–1176[Abstract/Free Full Text]
49. Schwartz BD, Zhu Y-S, Cordero J, Imperato-McGinley J, 5-Reductase deficiency and complete androgen insensitivity: natural models to suggest a direct role for androgens on bone density in men [Abstract OR23–1]. Proc of the 81st Annual Meet of The Endocrine Society, San Diego, CA, 1999

This article has been cited by other articles:

				A. E Lemus, J. Enriquez, A. Hernandez, R. Santillan, and G. Perez-Palacios Bioconversion of norethisterone, a progesterone receptor agonist into estrogen receptor agonists in osteoblastic cells J. Endocrinol., February 1, 2009; 200(2): 199 - 206. [Abstract] [Full Text] [PDF]

				S. J. Jacobsen, T. C. Cheetham, R. Haque, J. M. Shi, and R. K. Loo Association Between 5-{alpha} Reductase Inhibition and Risk of Hip Fracture JAMA, October 8, 2008; 300(14): 1660 - 1664. [Abstract] [Full Text] [PDF]

				J. Enriquez, A. E. Lemus, J. Chimal-Monroy, H. Arzate, G. A Garcia, B. Herrero, F. Larrea, and G. Perez-Palacios The effects of synthetic 19-norprogestins on osteoblastic cell function are mediated by their non-phenolic reduced metabolites J. Endocrinol., June 1, 2007; 193(3): 493 - 504. [Abstract] [Full Text] [PDF]

				M. T Zarrabeitia, J. L Hernandez, C. Valero, A. Zarrabeitia, J. A Amado, J. Gonzalez-Macias, and J. A Riancho Adiposity, estradiol, and genetic variants of steroid-metabolizing enzymes as determinants of bone mineral density Eur. J. Endocrinol., January 1, 2007; 156(1): 117 - 122. [Abstract] [Full Text] [PDF]

				A. M. Pino, J. M. Rodriguez, S. Rios, P. Astudillo, L. Leiva, G. Seitz, M. Fernandez, and J P. Rodriguez Aromatase activity of human mesenchymal stem cells is stimulated by early differentiation, vitamin D and leptin J. Endocrinol., December 1, 2006; 191(3): 715 - 725. [Abstract] [Full Text] [PDF]

				V. Sobel, B. Schwartz, Y.-S. Zhu, J. J. Cordero, and J. Imperato-McGinley Bone Mineral Density in the Complete Androgen Insensitivity and 5{alpha}-Reductase-2 Deficiency Syndromes J. Clin. Endocrinol. Metab., August 1, 2006; 91(8): 3017 - 3023. [Abstract] [Full Text] [PDF]

				M. Muir, G. Romalo, L. Wolf, W. Elger, and H.-U. Schweikert Estrone Sulfate Is a Major Source of Local Estrogen Formation in Human Bone J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4685 - 4692. [Abstract] [Full Text] [PDF]

				D. Vanderschueren, L. Vandenput, S. Boonen, M. K. Lindberg, R. Bouillon, and C. Ohlsson Androgens and Bone Endocr. Rev., June 1, 2004; 25(3): 389 - 425. [Abstract] [Full Text] [PDF]

				R. A. Anderson, A. M. Wallace, N. Sattar, N. Kumar, and K. Sundaram Evidence for Tissue Selectivity of the Synthetic Androgen 7{alpha}-Methyl-19-Nortestosterone in Hypogonadal Men J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2784 - 2793. [Abstract] [Full Text] [PDF]

				J. Compston Local Biosynthesis of Sex Steroids in Bone J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5398 - 5400. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Issa, S. Articles by Schweikert, H.-U. Search for Related Content PubMed PubMed Citation Articles by Issa, S. Articles by Schweikert, H.-U.