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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 9 4685-4692
Copyright © 2004 by The Endocrine Society

Estrone Sulfate Is a Major Source of Local Estrogen Formation in Human Bone

Mischa Muir, Gabriela Romalo, Lutz Wolf, Walter Elger and Hans-Udo Schweikert

Department of Internal Medicine (M.M., G.R., H.-U.S.), University of Bonn, 53111 Bonn, Germany; Department of Orthopedics (L.W.), St. Petrus-Krankenhaus, 53113 Bonn, Germany; and EnTec (W.E.), 07745 Jena, Germany

Address all correspondence and requests for reprints to: Hans-Udo Schweikert, M.D., Department of Internal Medicine, Division of Endocrinology, University of Bonn, Wilhelmstrasse 35-37, 53111 Bonn, Germany. E-mail: h.u.schweikert{at}uni-bonn.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrone sulfate (E1S) is the most abundant estrogen in the circulation of adults. The present study was undertaken to assess estrone (E1) and estradiol formation from E1S in freshly resected bone [bone fragments (BFs)] and osteoblast-like cells (hOB) cultured from BFs. Furthermore, we compared estrogen formation from E1S in rat and human osteosarcoma (OS) cell lines and that of estrogen formation from E1S with that of aromatization of androstenedione and testosterone in BFs and those from E1S and androstenedione in hOB cells. The bone used was from the head of the femur from a total of 15 women and 12 men. Steroid sulfatase activity (STA) was found, and the formation of estrone and estradiol from E1S was demonstrated. STA was similar in cells derived from BFs of men and women. STA was significantly lower in OS cell lines, compared with hOB cells. Estrogen formation from E1S in BFs was at least 20 times higher than that from androstenedione and about 50 times higher than that from testosterone. Similarly, estrogen formation from E1S in hOB cells exceeded the values derived from aromatization of androstenedione by two orders of magnitude. Based on these results, we conclude that hOB cells express the same pattern of E1S metabolism as resected bone and thus may accurately mirror the in vivo situation in man. In comparison with hOB cells, STA is fundamentally lower in widely used OS cell lines that express an osteoblastic phenotype. This shortcoming precludes their use as model cell lines to unravel STA metabolic pathways and its regulation in nontumorous bone. E1S is a major source of local bioactive estrogen formation in human bone. Because bone is highly susceptible to estrogen action, local estrogen formation from E1S may play an important role in bone maturation and homeostasis, particularly in elderly adults.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CIRCULATING ESTRADIOL IS an important factor in determining bone turnover and bone density in postmenopausal women and aging men (1, 2, 3, 4, 5, 6). Estrone sulfate (E1S) is an abundant estrogen precursor in the circulation of men, nonpregnant women, and postmenopausal women. E1S levels in postmenopausal and premenopausal women are several fold higher than those of unconjugated estrogens, and the same is true for men (7, 8, 9, 10). Because estrogens exert major beneficial effects on bone of both women and men and no systematic studies are available on free estrogen formation from E1S in mature human bone, we investigated whether resected human bone [bone fragments (BFs)] and first-passage osteoblast-like (hOB) cells cultured from BFs express steroid sulfatase (STS), which is required for the formation of estrone (E1) and, after further metabolism, for estradiol-17ß (E2). In addition, estrogen formation from E1S was compared in hOB cell strains and osteosarcoma (OS) cell lines. Furthermore, rates of estrogen formation from E1S, androstenedione, and testosterone were compared in BFs and those from E1S and androstenedione in hOB cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Silica gel thin-layer chromatography sheets with a plastic back (Polygram Sil G-Hy) were obtained from Machery and Nagel (Düren, Germany). [6,7-3H]Estrone sulfate (53 Ci/mmol), [4-14C]estrone (56.6 mCi/mmol), [4-14C]estradiol-17ß (53 mCi/mmol), and [1ß-3H]androstenedione (23.1 Ci/mmol) were purchased from Perkin-Elmer NEN Life Science Products (Brussels, Belgium). Nonradioactive steroids (E1, estradiol, dexamethasone) were purchased from Steraloids (Paesel, Frankfurt, Germany). The STA inhibitor estrone sulfamate was obtained from Sigma (Deisenhofen, Germany). MEM supplemented with Earl’s salts and L-glutamine (MEM), McCoy’s 5A medium plus Glutamax-I (L-alanyl-L-glutamine, a stabilized derivate of L-glutamine), DMEM plus Glutamax-I, penicillin, streptomycin, and nonessential amino acids were obtained from Life Technologies, Inc. (Karlsruhe, Germany). Dulbecco’s PBS was purchased from Biochrom (Berlin, Germany). Fetal calf serum (FCS) was from Cytogen (Ober-Mörlen, Germany). Tissue culture dishes (Falcon Primaria, 60 x 15 mm; polystyrole with a modified surface) were obtained from Becton Dickinson (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 reverse transcriptase, 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 used as supplied by the manufacturer.

Source and preparation of tissue

The bone used in this study was from the head of the femur, which was removed at orthopedic surgery from 15 women (age range 43–80, 66.7 ± 11.2 yr, mean ± SD) and 12 men (age range 47–85, 70.3 ± 6.9 yr) undergoing total hip replacement because of 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 the University of Bonn.

After resection, the bone was immediately placed in ice-cold saline. First, a biopsy was taken for later histological analysis, which revealed no inflammation, malignancy, or necrosis. The cancellous bone was then removed, thoroughly cleaned of blood and bone marrow, ground, and dissected into fine fragments as described (11, 12, 13, 14). The bone was then used for either immediate incubation and analysis of [3H]E1S metabolism or cell culture to obtain hOB cells.

Cell culture and phenotypic characteristics of cells

hOB cells. The methods used for hOB cell culture and the phenotypic characterization of the cells were as reported before (11, 14). The cultured bone cells displayed typical osteoblastic features of differentiated osteoblasts 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. Cellular proliferation normally occurred within 5–7 d of plating, and cultures in general reached confluence after 4–6 wk post plating. In all experiments, hOB cells were used in the first passage. Growth and phenotypic characteristics of the hOB cells compare well with those described in detail by the laboratory of Gallagher et al. (15).

OS cell lines. Three human (MG-63, HTB-96, CRL-1543) and three rat OS cell lines (CRL-1663, CRL-1661, ROS 17/2.8) were used. ROS 17/2.8 cells were a gift from Dr. R. Ziegler (Department of Internal Medicine, University of Heidelberg, Heidelberg, Germany), MG-63 was obtained from Flow Laboratories (Meckenheim, Germany), and all other cell lines were purchased from the American Type Culture Collection (Manassas, VA). The cell line MG-63 was derived from an OS of a 14-yr-old boy. It was found to be a good human osteoblast-like cell model to study the modulation of a number of the features of the osteoblast-like phenotype (16). CRL-1543 and HTB-96 were from an OS of a 15- and 13-yr-old girl, respectively . The rat OS cell lines CRL-1663 and CRL-1661 are cloned derivatives of a transplantable rat OS. According to American Type Culture Collection, the PTH responsiveness of cell line CRL-1661 was found to be higher than that of CRL-1663. ROS 17/2.8 is a clonal OS cell line with several osteoblastic characteristics that was derived from a transplantable rat OS (17). HTB-96 was cultured in McCoy’s 5A medium plus Glutamax-I, ROS 17/2.8 in MEM, CRL-1543, CRL-1661, CRL-1663, and MG-63 in DMEM plus Glutamax-I. All media were enriched with 10% FCS.

Determination of free steroid formation from [3H]E1S in BFs, hOB cell strains, and OS cell lines

BFs. BFs (250–300 mg) were transferred to 16 x 100 mm stopper-capped culture tubes and incubated with a mixture consisting of Krebs-Ringer phosphate buffer (pH 7.4), glucose (1 mM), and [3H]E1S (at varying concentrations, depending on the type of experiment) in a total volume of 1 ml. After capping the tubes, the mixtures were incubated at 37 C under air for varying periods of time. The reactions were stopped by chilling in crushed ice. Determination of free estrogen formation was performed essentially as described by Milewich et al. (18). In brief, 0.8-ml aliquots of incubation media were transferred to 16 x 100 mm stopper-capped tubes. To each sample [4-14C]E1 (11,000 dpm) and [4-14C]E2 (11,000 dpm) were added as internal recovery standards, followed by addition of 4.0 ml chloroform and 0.4 ml water. The tubes were capped, and the mixtures were vortexed three times for 10 sec during a 10-min period. The samples were centrifuged (2000 rpm) at 4 C, and the chloroform layers were transferred to 25 x 150 mm stopper-capped tubes. The aqueous phases were reextracted four times with 4 ml chloroform, and the pooled chloroform extracts were backwashed five times with 4 ml water. STA was then determined in a 0.1-ml aliquot (determination of hydrolysis). A second 0.5-ml aliquot to which 25 µl of a carrier mixture containing 25 µg each of E1 and E2 had been added was used for product separation and quantification. For this purpose, the samples were dried, redissolved in 50 µl chloroform and spotted on thin-layer plates. This procedure was repeated once more. The steroids were separated using the solvent system toluene/ethanol (85:15; vol/vol), and [3H]E1 and [3H]E2 formed were quantified as described to determine STA (18).

HOB cell strains and OS cell lines. Cells were obtained and cultured as described above. When cells reached confluence, the medium was removed, the cells were washed with PBS (2 ml), and thereafter they were incubated for 24 h with the corresponding medium in the absence of FCS. This medium was then replaced with 1 ml MEM containing [3H]E1S in various concentrations. In general, cell incubations were for 6 h and were conducted in a humidified incubator at 37 C in the presence of 5% CO2. The enzymatic reactions were stopped by placing the dishes on crushed ice. Medium was transferred to 16 x 100 mm stopper-capped tubes and chloroform (5 ml) was added. STA and the formation of [3H]E1 and [3H]E2 were then determined as described above for ground bone. The formation of [3H]E1 recovered after the incubation of four different hOB cell strains with 0.05 µM [3H]E1S was confirmed after addition of [14C]E1 to the incubation medium followed by chromatography and recrystallization to constant specific activity and constant 3H/14C ratios essentially as described previously (19, 20) (data not shown).

Assays of aromatase activity

Two different assays for determination of aromatase activity (AA) were used essentially as described previously (12, 19, 20). Estrogen formation (aromatization) from [3H]androstenedione and [3H]testosterone, respectively, in BFs was assessed by measurement of tritiated E1 and E2 formation from [1,2,6,7-3H]androstenedione and [1,2,6,7-3H]testosterone, respectively, and/or by determination of [3H]H2O generation from [1ß-3H] androstenedione or [1ß-3H] testosterone. In confluent monolayers of hOB cells, AA was determined after preincubation for 24 h with either dexamethasone (dissolved in 15 µl ethanol and added to the medium to give a final concentration of 0.01 µM) or without dexamethasone but with 15 µl solvent. Cells were then incubated with 0.05 µM [1ß-3H]androstenedione for various periods of time either with or without 0.01 µM dexamethasone.

Protein determination

Cell protein was determined by the method of Lowry et al. (21).

mRNA assessment

Total RNA was isolated from hOB cells and OS cell lines 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) containing 25 mM Tris-HCl, 5 mM MgCl2, 0.1 mM EDTA, and 2 U RNase-free deoxyribonuclease I per microgram 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 dATP, dCTP, dGTP, and 2'deoxythymidine 5'triphosphate; 0.5 µg oligo dT12–18-primer; 1 µg total RNA; and 200 U Superscript II reverse transcriptase. Mixtures were incubated at 42 C for 55 min and then stored at –20 C.

A 448-bp fragment of the human steroid sulfatase (STS) cDNA was amplified by nested PCR using the following two pairs of oligonucleotides designed according to the published sequence (EMBL Data Bank: M16505): first reaction, STS forward, 5'-GGAGATGCCTTTAAGGAAGAT-3' (sense, position 217–237) and STS reverse, 5'-CTCTTGAAGCCCGTGGTGAA-3' (antisense, position 752–771); and nested reaction, 5'-GAGAGCCACGAAGCATCAA-3' (sense, position 278–296) and 5'-CTCTCAGATTGGTCAAAGAGA-3' (antisense, position 705–725).

PCRs were performed using the 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 for first reaction; 2 µl cDNA; and 0.5 U Taq DNA polymerase. Cycling consisted of an initial denaturation (5 min at 94 C), followed by 35 cycles of denaturation (40 sec at 94 C), annealing (30 sec at 60 C), and polymerization (45 sec at 72 C) and one final elongation step (7 min at 72 C). The products (2 µl of a 1:5 dilution) were subjected to the nested reactions using identical reaction mixtures except for the STS forward nested and STS reverse nested oligonucleotides (6 pmol each). Cycling conditions were the same as described above.

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

Statistical analysis

Student’s t test was used for statistical analysis of the data. The calculations were performed using Origin version 7 software (OriginLab Corp., Northampton, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BFs

After incubation of BFs with [3H]E1S, the formation of [3H]E1 and [3H]E2 was detected in all experiments, thus demonstrating that both STS and 17ß-hydroxysteroid oxidoreductase are expressed in this tissue.

The time course of the appearance of [3H]E1 and [3H]E2 formed after incubation of [3H]E1S with bone obtained from three patients was studied in the absence of added cofactors. In each instance, as depicted in Fig. 1Go, [3H]E1 formation was linear with time for approximately 90 min and plateaued thereafter. Although the major metabolite formed was [3H]E1, in some instances after 60 min trace amounts of [3H]E2 were also measurable. STA as determined in bone from four donors using [3H]E1S concentrations of 0.05 µM was 0.75 ± 0.18 pmol·100 mg bone–1·h–1 (mean ± SEM).



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  		FIG. 1. Time course of [3H]E1S metabolism by human bone. Each sample contained thoroughly cleaned BFs (200–300 mg) obtained from the head of the femur of two women, 72 (-•-) and 80 (-{blacktriangleup}-) yr of age, and from a 74-yr-old man (-{blacksquare}-). The samples were incubated in Krebs-Ringer phosphate buffer (pH 7.4) containing [3H]E1S (0.05 µM) and glucose (5 mM) in a total volume of 1 ml. At the indicated intervals, the reaction was terminated and the STA was assessed in duplicate samples.

 
When STA was determined as the relationship between [3H]E1S concentration (0.05–15 µM) and estrogen formation, estrogen formation increased until a plateau was approached at approximately 15 µM [3H]E1S (Fig. 2Go). E1 was the major metabolite formed. As estimated from the double reciprocal plot, the apparent Michaelis constant (Km) of bone sulfatase was approximately 5.2 µM, and the apparent maximum rate of estrogen formation was 44 pmol·100 mg bone–1·h–1.



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  		FIG. 2. STA in human bone as a function of [3H]E1S concentration. Samples containing bone (BFs, approximately 250 mg) from a 62-yr-old woman were incubated for 90 min with [3H]E1S in concentrations ranging from 0.05 to 15 µM. STA was then assessed. A double reciprocal plot of the data is shown in the inset.

 
[3H]E1S hydrolysis was nearly completely blocked when incubations of BFs were conducted in the presence of estrone sulfamate (1 µM), a specific STS inhibitor (22).

hOB cell strains and OS cell lines

As with freshly removed bone, the studies of [3H]E1S metabolism by hOB cells were conducted under standard conditions. The time course of [3H]E1S hydrolysis and the appearance of [3H]E1 and [3H]E2 after incubation for 2–48 h was studied in two cell strains. As illustrated in Fig. 3Go, hydrolysis of [3H]E1S and formation of [3H]E1 increased linearly for approximately 6 h. There was a linear increase in [3H]E2 formation starting after 6 h.



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  		FIG. 3. Time course of the metabolism of [3H]E1S in hOB cell strains. Cells from bone of a 49-yr-old woman and a 72-yr-old man were grown as described in Materials and Methods. When the cells were confluent, the medium was replaced with fresh MEM containing [3H]E1S (0.05 µM). At the indicated times, cells were harvested and the formation of E1 and E2 (woman: E1: -•-, E2: -{circ}-; man: E1, -{blacksquare}-, E2: -{square}-) was assessed as described in the text. In all instances product formation was determined in duplicate samples.

 
The relationship between the concentration of [3H]E1S and STA was assessed in nine different hOB cell strains, obtained from five women and four men, using [3H]E1S at concentrations of 0.05–20 µM. As presented in Fig. 4AGo, STA increased until a plateau was approached at approximately 15 µM, and again [3H]E1 was the major metabolite formed. The apparent Km of sulfatase as estimated from the Lineweaver-Burk plot was 5.9 ± 1.6 µM (mean ± SEM), and the apparent maximal velocity was 853 ± 296 pmol·mg protein–1·h–1. The Eadie-Scatchard plot of the data, as presented in Fig. 4BGo, indicated nonlinear rather than linear kinetics, which might suggest the presence of more than one sulfatase isozyme in bone.



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  		FIG. 4. STA in hOB cell strains at increasing [3H]E1S concentrations. A, Cells from bone of five women and four men were grown in standard medium, as described in the text. When the cells became confluent, the medium was replaced with MEM containing [3H]E1S at the indicated concentrations (0.05–20 µM). After 6 h, the medium was removed for determination of STA, and the cells were harvested for protein determination. In all instances STA was determined in duplicate samples and is expressed as picomoles per milligram protein–1 per hour–1. Inset, Double reciprocal transformation of data (Lineweaver-Burk plot). B, Eadie-Scatchard plot of the data presented in A. V, Velocity of sulfatase activity (picomoles·milligrams protein–1·hours–1; S, substrate concentration (micromoles).

 
STA was assessed next in a hOB cell strain in the presence of estrone sulfamate (0.1 nM to 1 µM). The inhibitor efficiently blocked STA with an estimated IC50 of approximately 0.3 nM (data not shown). Having defined the standard incubation conditions, STA was then assessed in hOB cell strains cultured from 12 women (age range 49–80, 68.8 ± 9.4 yr, mean ± SD) and five men (age range 65–85, 73.4 ± 7.5 yr, mean ± SD). Cells were incubated with 0.005 µM [3H]E1S, a concentration that is within the normal range found in human adults (10). STA was detected in all cell strains examined with similar values in both sexes (women: 1.04 ± 0.14; mean ± SEM; men: 1.29 ± 0.41 pmol·mg protein–1·h–1).

Next, STA of hOB cells and from each of three different human and rat OS cell lines was compared in confluent monolayers. STA in the OS cell lines was measured in four different experiments. Cells were incubated with 0.05 µM [3H]E1S for 6 h. As illustrated in Fig. 5Go, STA was significantly lower (P < 0.02) in all OS cell lines, compared with 15 different hOB cell strains that were incubated under identical conditions. To confirm these biochemical results, RT-PCRs demonstrated the expression of STS mRNA in hOB cells and OS cell lines. A cDNA product of the expected size as described in Materials and Methods was found in all of the hOB cell strains and OS cell lines examined.



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  		FIG. 5. Comparison of STA in hOB cell strains and OS cell lines. HOB cell strains derived from bone of 15 patients (seven women and eight men) and in three human and three rat OS cell lines were grown as described in Materials and Methods. One hOB cell strain was used in two different experiments. When the cells reached confluence, the medium was replaced for 24 h with corresponding medium without FCS and thereafter with MEM containing [3H]E1S (0.05 µM). After 6 h, the cells were harvested for protein determination, and STA was determined as described in the text. In all instances STA was determined in duplicate samples. STA of OS cell lines was assessed in four independent experiments. Shaded bars, human OS cell lines; black bars, rat OS cell lines. Degree of significance of hOB cell strains vs. OS cell lines: **, P < 0.002; *, P < 0.02.

 
Estrogen formation from E1S and androstenedione (each at a concentration of 0.05 µM, in the presence or absence of 0.01 µM dexamethasone) was determined in parallel experiments in two hOB cell strains for incubation periods varying from 2 to 24 h. As shown in Fig. 6Go, AA was very low to undetectable in the absence of dexamethasone but was stimulated markedly by dexamethasone, whereas STA remained unaffected. In every instance, STA exceeded that of dexamethasone-stimulated AA by 2 orders of magnitude.



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  		FIG. 6. Comparison of the rates of STA (A) and AA (B) in two different hOB cell strains. Confluent hOB cell monolayers were preincubated for 24 h in serum-free medium with or without 0.01 µM dexamethasone. The cells were then incubated in parallel experiments with either 0.05 µM [3H]estrone sulfate or 0.05 µM [1ß-3H]androstenedione. Activities were determined as described in the text. All samples were incubated in duplicate, and each symbol represents the mean value. Closed symbols, enzyme activity measured in the presence, open symbols, in the absence of dexamethasone., •, {circ}, osteoblasts grown from bone of a 73-yr-old woman; {blacktriangleup}, {triangleup}, hOB cells derived from bone of a 76-yr-old man.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study is part of ongoing investigations in our laboratory aimed at understanding how gonadal steroids exert their osteoprotective effects in human bone. Previously we demonstrated that androstenedione and testosterone serve as substrates for the formation of estrogen in bone and cultured hOB cells obtained from men and women (12, 13, 23). Because bone and cultured hOB cells express estrogen receptors (24, 25), it is reasonable to assume that locally formed estrogen acts directly at the site of biosynthesis. STA has been demonstrated in murine and human OS cell lines and fetal hOB cells (26, 27, 28); however, systematic studies on this enzyme in mature human bone cells have hitherto been lacking. Thus, the metabolism of [3H]E1S was examined in BFs and in first-passage human osteoblasts cultured from bone. We demonstrated that bone contains the enzymatic machinery required to form E1 and E2 from E1S, a major estrogen precursor in circulation. Further systematic studies in differentiated cultured osteoblasts showed that hOB cells mirrored the enzyme pattern found in bone. STA in human and rat OS cell lines is significantly lower than in hOB cells. Finally, local estrogen formation from E1S was shown to exceed that derived from aromatization by 1–2 orders of magnitude.

Based on these findings, we draw the following conclusions: mature bone and hOB cells can convert biological inactive E1S, present in serum of men and nonpregnant women in relatively high concentrations, into free E1, which subsequently is metabolized by 17ß-hydroxysteroid oxidoreductase action to the biologically potent estrogen E2. It is of interest to note that first-passage cultured hOB cells express the same pattern of steroid metabolism as freshly resected bone and thus appear to accurately reflect the in vivo situation in humans.

It is now well documented that estrogen plays a dominant role in bone maturation and bone mass maintenance. Data obtained from human mutations impairing either estrogen action or formation and from corresponding knockout mouse models show that in the male, estrogen plays a major role in the control of bone growth as well as in the accumulation and maintenance of bone mass (29, 30). It is thus conceivable that both circulating estrogen and local estrogen formation in bone arising from aromatization of circulating androgens and free estrogen formation from E1S in the growing and mature skeleton might be important sources of estrogen.

Local estrogen formation might have an even higher impact on the development of osteoporosis in the elderly. There is now abundant evidence that loss of bone mineral density is directly related to declining estrogen levels in both men and women with advancing age (1, 2, 3, 4, 5, 6, 31, 32). However, it remains enigmatic as to why only a fraction of peri- and postmenopausal women develop fast bone loss, although all are estrogen deficient. One possible explanation is that the levels of E1S and other estrogen precursors in peri- and postmenopausal women who develop fast bone loss are lower than in those that do not, thus providing less substrate to bone cells for the formation of biologically active estrogens. Because the apparent Km value of bone sulfatase exceeds by far the serum E1S levels, it is conceivable that the formation of free estrogen from this substrate under physiological conditions is mainly a function of substrate availability. This view is substantiated by a recent study showing lower serum E1S levels in osteoporotic women with greater loss in femoral neck bone mineral density (fast losers), compared with non losers (5). Furthermore, our data, which did not reveal a significant difference of STA in hOB cells from women and men of different ages exposed to a fixed physiological concentration of 0.005 µM E1S, support this assumption. It is also possible that serum estrogen levels underestimate the osteoprotective effect of local estrogen synthesis (32) and that either E1S uptake into bone cells or local STA might decline with age.

E2 formation from E1, the direct product of E1S hydrolysis, in bone might also be rate limiting. Previously we assessed the expression of 17ß-hydroxysteroid oxidoreductase isozymes in hOB cells and were able to demonstrate the presence of the isozymes types 1–4 (33). Although we have not performed quantitative PCR, it appears likely that the 17ß-hydroxysteroid oxidoreductase isozymes types 3 and 4 in hOB cells are predominant because the expression of the types 1 and 2 isozymes could be detected using only nested PCR. In addition, we found that in mature hOB cells, the formation of E2 from E1 (activation) rather than E1 formation from E2 is the preferred metabolic pathway (34). It is therefore conceivable that the amount of locally formed E2 from E1 also plays an important osteoprotective role in this tissue.

Studies using nine different hOB cell strains and E1S concentrations ranging from 0.05 to 20 µM indicated nonlinear rather than linear enzyme kinetics. This behavior suggests the presence of more than one sulfatase. It is of interest that Shankaran et al. (35) reported two pH optima for STS in human placenta, which supports the presence of sulfatase isozymes. Several sulfatase genes (Xp22.3 sulfatases) have been located on the short arm of the human X chromosome, which include steroid aryl sulfatase and three newly identified members of the sulfatase gene family (36). It is therefore possible that hOB cells harbor more than one STS isozyme.

STA was significantly lower in OS cell lines, derived from both human and rat osteosarcomas, compared with that in hOB cells. The physiological significance of this finding at present remains unclear; however, it is evident that widely used OS cell lines (which display an osteoblastic phenotype) express a fundamentally different STA pattern in comparison with hOB cells. This difference precludes their use as model cell lines to unravel STA metabolic pathways and its regulation in nontumorous bone.

Compared with our previous results on the rate of aromatization of androstenedione (12) and testosterone (Ref.13 ; and Romalo, G., L. Wolf, and H. U. Schweikert, unpublished results) in freshly removed bone, the rate of estrogen formation from E1S in bone was at least 20 times higher than that from androstenedione and 50 times higher than that of testosterone. In these former studies, we found mean rates of estrogen formation from 0.05 µM androstenedione of 0.041 ± 0.0075 (n = 15) and 0.016 ± 0.0046 pmol·100 mg dry bone weight–1·h–1 from 0.05 µM testosterone (n = 11). In the present study, the mean rate of estrogen formation from [3H]E1S at a concentration of 0.05 µM was 0.75 ± 0.18 pmol·100 mg wet bone weight–1·h–1. Similarly, estrogen formation from E1S in hOB cells exceeded the values derived from aromatization of androstenedione by 2 orders of magnitude.

The results of our experiments on estrogen formation from androstenedione and testosterone in bone compare well with in vivo data of MacDonald and coworkers (37, 38), who reported that androstenedione in women and men is a better substrate for peripheral aromatization than testosterone. In women testosterone did not serve as a significant circulating precursor for peripheral estrogen formation because serum testosterone levels are low and the steroid was inefficiently converted to estradiol, and in men the peripheral conversion rates of androstenedione to estrone exceeded those of testosterone to estradiol by about 3- to 6-fold.

In conclusion, we have demonstrated that both freshly resected human bone as well as hOB cells can effectively form E1 and E2 from circulating E1S and that E1S is a major source of local bioactive estrogen formation in the human bone. Because local regulation of hormone activity in target tissues offers obvious favorable effects (39), such as providing mechanisms for tissue-specific responses when systemic hormone production remains unchanged and for preservation of homeostasis in the presence of alterations in hormonal status, it is possible that estrogen formation from E1S plays an important role in steroid hormone action in mature bone, a tissue that is highly susceptible to estrogen action.


   Acknowledgments
 
We thank Dr. H. E. Schaefer (Department of Pathology, University of Freiburg, Freiburg, Germany) for histological analysis of bone specimens, Dr. Maritta Feix (Department of Internal Medicine, University of Bonn, Bonn, Germany) for the sulfatase-mRNA analysis, and Drs. Leon Milewich (Department of Obstetrics and Gynecology, The University of Texas, Southwestern Medical Center, Dallas, TX) and David W. Russell (Department of Molecular Genetics, The University of Texas, Southwestern Medical Center) for suggestions and critical reading of the manuscript. Mrs. Margarete Sudmann and Mrs. Maria Hardt provided able technical assistance in the performance of these studies.


   Footnotes
 
This work was supported by a grant from the Bundesministerium für Bildung und Forschung.

Results from this work were presented in part at the 82nd Annual Meeting of The Endocrine Society, 2000, Toronto, Canada.

Abbreviations: AA, Aromatase activity; BF, bone fragment; E1, estrone; E2, estradiol-17ß; E1S, estrone sulfate; FCS, fetal calf serum; hOB, osteoblast-like cell; Km, Michaelis constant; OS, osteosarcoma; STA, STS activity; STS, steroid sulfatase.

Received January 14, 2004.

Accepted May 25, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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