The role of estrogens in men and androgens in women

The role of estrogens in men and androgens in women

Endocrinol Metab Clin N Am 32 (2003) 195–218 The role of estrogens in men and androgens in women Sundeep Khosla, MDa,*, John P. Bilezikian, MDb a Di...

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Endocrinol Metab Clin N Am 32 (2003) 195–218

The role of estrogens in men and androgens in women Sundeep Khosla, MDa,*, John P. Bilezikian, MDb a

Division of Endocrinology, Metabolism, and Nutrition, Mayo Clinic and Foundation, 200 First Street SW, 5-194 Joseph, Rochester, MN 55905, USA b Division of Endocrinology, Departments of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA

Sex steroids play a crucial role in the assembly and maintenance of the female and male skeletons. The most obvious clinical example of this is the accelerated bone loss that follows the development of estrogen deficiency in women after natural or surgical menopause [1]. It is now clear that estrogens and androgens are important for the acquisition and preservation of bone mass in women and in men [2]. Traditionally, it was generally believed that the role of estrogen and testosterone was gender specific. Thus, estrogen was believed to be critical for the female skeleton and testosterone for the male skeleton. It is now evident, however, that this gender specificity was, in fact, an unfortunate bias in our thinking. It seems that for bone (and likely for other tissues), nature intended important roles for each sex steroid in both genders. Thus, androgens likely have important effects on the female skeleton and, somewhat to the surprise of most of us, estrogen seems to play a dominant role in regulating bone metabolism in the male [2]. Convincing evidence for this gender ‘‘cross-talk’’ has come not only from animal and human studies, including several sentinel and highly informative ‘‘experiments of nature’’ [3–6], but also from studies in the laboratory demonstrating actions of estrogen by way of the androgen receptor and of androgens (and androgen metabolites) by way of the estrogen receptor [7,8]. This compelling evidence from multiple, independent lines of investigation, is now forcing a major reevaluation of our traditional notions of sex steroid

This work was supported by grants AG04875 and AR27065 from the National Institutes of Health (SK) and FDR 1024 (JPB). * Corresponding author. E-mail address: [email protected] (S. Khosla). 0889-8529/03/$ - see front matter Ó 2003, Elsevier Science (USA). All rights reserved. doi:10.1016/S0889-8529(02)00087-7

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action on bone that is likely also to impact on our concepts of sex steroid action in other tissues.

Role of estrogen in bone metabolism in men Human ‘‘experiments of nature’’ Alpha-estrogen receptor deficiency in men In 1994, Smith and associates [3] provided seminal information about the importance of the estrogen alpha receptor in male skeletal development. They described a 28-year-old man with a mutation in the alpha-estrogen receptor gene [3]. He was the product of a consanguineous marriage in which both parents carried a single copy of the abnormal estrogen alphareceptor gene. The point mutation was located at codon 157, where thymidine had replaced cytosine. The resulting stop codon was associated with an estrogen receptor that is severely truncated and cannot bind estrogen. The subject’s baseline estradiol (119 pg/ml) and estrone (145 pg/ml) levels were above normal. Although bound and free testosterone and dihydrotestosterone concentrations were normal, the concentrations of LH (37 mIU/ml) and FSH (33 mIU/ml) were in the mildly castrate range. He was extremely tall (204 cm) with eunuchoid proportions. The epiphyses were still open (bone age, 15 years) and he was still growing. Genu valgum was prominent in the lower extremities. There were no features of acromegaly. The growth curve was steady and continuous, without evidence for a pubertal growth spurt. As measured by dual energy x-ray absorptiometry, bone density of the lumbar spine was 0.745 g/cm2, corresponding to two standard deviations below average for a 15-year-old boy. He was given large doses of exogenous estrogen (transdermal ethinyl estradiol), with serum concentrations of estradiol reaching 10-fold higher than the typical male, 270 pg/ ml(nl: 10–50), but there was no response. Aromatase deficiency in men Theoretically, men can be rendered estrogen deficient if the aromatase enzyme responsible for the conversion of androgens to estrogens is defective or absent. Three men have been reported with aromatase deficiency caused by a genetic defect in the aromatase gene. Each, the product of a consanguineous marriage [4,5,9], shows a point mutation in exon IX [4,5] or at exon V [9] in the aromatase gene. Morishima et al [4] described a 24-year-old man with a cytosine to thymidine base pair change in exon IX at position 1123 and a resultant amino acid substitution at R375, adjacent to the heme binding site. Similarly, Carani et al [5] described a 31-year-old man with a single base pair change in exon IX at position 1094 (guanine to adenine) with the amino acid substitution at R365. In a newborn male, reported by Deladoey et al. [9], the mutation was found in exon V, causing a frame shift mutation with an ensuing premature stop codon. In each of these three

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Table 1 Biochemical parameters in a man with aromatase deficiency Calcium Phosphorus PTH 25-hydroxyvitamin D 1,25-dihydroxyvitamin D Alkaline phosphatase Urinary calcium Deoxypyridiniline

9.9 3.3 26 36 55 241 185 25.3

8.7–10.7 mg/dL 2.5–4.5 mg/dL 10–65 pg/mL 9–52 ng/mL 15–60 pg/mL 39–117 IU/L 150–300 mg/24 hours 4–19 nmol/mmol Cr

Data from Morishima A, Grumbach MM, Simpson ER, et al. Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. J Clin Endocrinol Metab 1995;80:3689–98.

cases, the gene product is completely inactive thus estrogen levels are not detectable. The two adult men reported by Morishima et al [4] and Carani et al [5] were tall with unfused epiphyses, a bone age of 15, eunuchoid features (upper segment/lower segment ¼ 0.85–0.88) and genu valgum. They were still growing. The patient of Morishima et al [4] had markedly elevated androgen levels. The complete biochemical profile of the patient described by Morishima et al [4] and Simpson et al [10] is shown in Tables 1 and 2. The growth curve of that patient, similar to the man with the alpha-estrogen receptor defect, did not give evidence for a pubertal growth spurt although in all other respects puberty was not delayed (Fig. 1). Both patients had reduced bone mass. The bone mineral density (BMD) of the patient described by Morishima et al [4] is shown in Fig. 2. Because of his enlarged areal density, a correction had to be applied to determine the bone mineral apparent density. Even more impressive reductions became evident when areal size was taken into account with T-scores of 1.99 (spine), 2.12 (femoral neck) and 7.75 (forearm). Both men with aromatase deficiency responded to the administration of estrogen [5,6]. The five-year follow-up data on the patient described by Morishima, Bilezikian and their colleagues [11] provide instructive commentary on the potential anabolic qualities of estrogens in the setting of

Table 2 Sex steroids and gonadotropin concentrations in a man with aromatase deficiency Estradiol Estrone Testosterone 5a-dihydrotestosterone FSH LH

\7 <7 2015 125 28.3 26.1

10–50 pg/mL 10–50 pg/mL 200–1200 ng/dL 30–85 ng/dL 5–9.9 mIU/mL 2.0–9.9 mIU/mL

Data from Simpson ER, Zhao Y, Agarwal VR, et al. Aromatase expression in health and disease. Recent Prog Horm Res 1997;52:185–214.

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Fig. 1. Growth curve and bone age before and after five years of estrogen therapy in a man with aromatase deficiency. After conjugated estrogen therapy was begun (bar), linear growth ceased promptly. Height has remained at 204 cm since therapy. All epiphyses were closed within six months (insets). The curves + and  numbers represent the mean and standard deviations for normal young men. (Reproduced from Bilezikian JP, Khosla S, Riggs BL. Estrogen effects on bone in the male skeleton. In Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of bone biology. San Diego: Academic Press; 2002. p. 1467–76; with permission).

the growing male skeleton. For most of the 5-year period of therapy, the dose of conjugated estrogen was 0.75 mg daily. On this dosage, estradiol levels were maintained within normal limits for men. Concomitant with return of estrogen levels to normal, androgen levels fell from markedly

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Fig. 2. Bone mineral density at baseline in a man with aromatase deficiency. For each site, the bone mass is shown as a two-dimensional value (g/cm2) and as bone mineral apparent density using correction factors for body size. For each site, estimated true bone density (g/cc) is substantially lower than the direct measurement, as shown in the shaded area of each bar. (Reproduced from Bilezikian JP, Khosla S, Riggs BL. Estrogen effects on bone in the male skeleton. In Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of bone biology. San Diego: Academic Press; 2002. p. 1467–76; with permission).

elevated levels to normal. The gonadotropins, LH and FSH, also returned to normal, illustrating the point that estrogens play an important role in controlling gonadotropin production in the male (Fig. 3). Within six months of beginning estrogen therapy, the patient reported by Bilezikian, Morishima et al [11], showed no further longitudinal growth and the epiphyses closed (Fig. 1). Bone mineral density increased dramatically. During the first three years, lumbar spine, femoral neck, and forearm improved by 20.7%, 15.7%, and 12.9% respectively. In years 4 and 5 of estrogen therapy, the gains in the lumbar spine and femoral neck were maintained with further marked increases in the forearm bone density, now 26% (Fig. 4). The effect of estrogen to improve bone mass in this setting is best described as anabolic because of the magnitude of the change and also because further bone growth did not occur. Without further bone growth, the change in BMD is likely to reflect improved mineralization per unit area. This property of estrogen to stimulate the acquisition of bone mass is different from its effects in the postmenopausal woman, in whom the estrogen effect is more accurately described as antiresorptive. In the syndromes of estrogen deficiency or resistance, height and inexorable growth, continuing well into adulthood, was not accompanied with a pubertal growth spurt. If estrogens are important for the pubertal growth

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Fig. 3. Changes in sex steroids, in gonadotropins, and in bone resorption with estrogen therapy in a man with aromatase deficiency. The data are shown for estradiol (A), testosterone (B), LH (C), and the resorption marker, deoxypyridinoline (D). Over 5 years, estradiol levels initially rose and then were maintained in the normal range for males when administered at 0.75 mg/day. Testosterone levels fell into the normal range as did LH and DPD levels. (Reproduced from Bilezikian JP, Khosla S, Riggs BL. Estrogen effects on bone in the male skeleton. In Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of bone biology. San Diego: Academic Press; 2002. p. 1467–76; with permission).

spurt, syndromes of estrogen excess might be expected to be linked to a premature growth spurt. Indeed, in the syndrome of aromatase excess, because of an activating mutation of the aromatase gene and elevated estrogen levels, puberty does occur prematurely [12–15]. In the testicular feminization syndrome, XY males do not respond to androgens because of a mutation in the androgen receptor but they respond normally to estrogens. Again, confirming an important role for estrogens in the male, the pubertal growth spurt is seen in these XY males [16,17]. Finally, premature skeletal maturation has been reported in estrogen-secreting tumors [18–20]. In the aggregate, these observations make a compelling point that in the male, as in the female, the pubertal growth spurt is a function of estrogens, not androgens. Animal ‘‘knockouts’’ of the estrogen receptor and aromatase genes Genetic models in which specific genes for the alpha estrogen receptor or the beta estrogen receptor are knocked out singly or together, and models in

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Fig. 4. Changes in bone mineral density with estrogen therapy in a man with aromatase deficiency. Percentage change is shown for lumbar spine (1), femoral neck (2), and distal radius (3). The data shown are not corrected for bone size. (Reproduced from Bilezikian JP, Khosla S, Riggs BL. Estrogen effects on bone in the male skeleton. In Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of bone biology. San Diego: Academic Press; 2002. p. 1467–76; with permission).

which the aromatase gene has been knocked out, have been helpful in elucidating further aspects of the role of estrogens in male skeletal development [21–23]. There are discrepancies between the genetic mouse models and the human gene ‘‘knock out’’ experiments of nature described above [24]. Mouse models are not as ‘‘clean’’ as human models. The phenotypes of mice rendered deficient in the alpha or beta estrogen receptor or deficient in aromatase activity do not invariably mirror overt human phenotypes. For example, the lack of the alpha estrogen receptor or the aromatase gene in human subjects is associated with continuous longitudinal bone growth. In the animal models, on the other hand, defects in these same genes are associated with reduced long bone growth. Moreover, it is not clear whether estrogen deficiency in the animal models is associated with high or low bone turnover, whereas in the human subjects with these gene defects, high bone turnover is consistently observed. Nevertheless, the animal gene knockouts have been instructive in further understanding the role of estrogens in male skeletal development. The alpha-estrogen receptor knockout mouse displays a 20%–25% reduction in bone density [21,25,26], consistent with the human patient described by Smith et al [3]. In contrast, the beta-estrogen receptor knockout male mouse shows no skeletal abnormality [27]. The same betaestrogen receptor defect in the adult female mouse, however, does show

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changes with an increase in bone mineral content caused by an increase in cross-sectional cortical area. Vidal et al [28] have characterized male mice deficient in the alpha estrogen receptor (ERKO) or the beta estrogen receptor (BERKO) or both (DERKO), helping to establish further a role for specific estrogen receptors in the acquisition of skeletal mass in the male. The importance of the alpha estrogen receptor in several skeletal characteristics of the male was clearly shown. Foreshortened long bone growth was seen only in the ERKO and DERKO mice but not in the BERKO animals, clearly attributing this estrogenic function to the alpha estrogen receptor, not the beta estrogen receptor. In addition, significant reductions in mice lacking the alpha estrogen receptor, not the beta receptor, were observed in total body mineral content, femoral bone mineral content, and spine bone mineral content. The abnormalities persisted in the ERKO and DERKO mice when animal weight and skeletal length were accounted for. On the other hand, cancellous bone density as analyzed by peripheral quantitative tomography (pQCT) and by bone histomorphometry was unchanged in all animals. Cortical bone mineral content by pQCT was diminished in ERKO and DERKO animals as compared with wild type. The reduction in cortical bone mineral content was caused by reduced cross-sectional area primarily because of smaller periosteal and endosteal circumferences. Similar to cancellous bone density, cortical volumetric density was unchanged. Mechanical strength was diminished in the ERKO animals with a tendency for a similar decline in the DERKO animals. Observations with these knockout mice clearly established differences in the alpha and beta estrogen receptors, with all abnormalities observed being clearly a function of the alpha receptor, not the beta receptor. In several key respects, the knockout animals did not confirm expectations that cancellous or cortical bone density would be diminished in the alpha estrogen receptor deficient knockout animal. These points are important but not clearly understood. It could be that the estrogen receptors are just not important in these aspects of the male mouse skeleton. Alternatively, it is possible that compensatory mechanisms were induced to adjust for the receptor deficiency. Estrogen and testosterone levels are elevated in these animals. Elevated levels could have accounted for the lack of some of these expected changes. It is even possible that the elevated estrogens in these knockout animals were somehow activating a truncated version of the alpha receptor that has been shown to be present in alpha-receptor knockout animals [29]. Consistent with this point, Gentile et al [30] have shown that in DERKO animals, estrogens can rescue the loss in bone density after ovariectomy when high levels of estrogens are used. Sims et al [31] have recently addressed this issue by creating male (and female) mice in which the gene knockouts are full and therefore do not express a truncated form of the receptor that theoretically could bind estrogen. Consistent with the report of Vidal et al [28] only the alpha

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estrogen receptor was shown to regulate bone remodeling in the male mouse. Lindberg et al [32] have continued their study of the relative importance of the alpha and beta estrogen receptor on the male mouse skeleton by taking a different tact. Recognizing that the compensatory adjustments in sex steroids caused by estrogen receptor deficiency may have masked observations that might otherwise have been made, they subjected wild type and the same knockout animals, ERKO, BERKO, and DERKO to orchidectomy at 7 months of age. Their experimental premise was that elimination of all sex steroids by orchidectomy would permit assignment of a specific role for estrogens when 17b-estradiol was used subsequently to prevent the abnormalities induced by the abrupt loss of androgens. After orchidectomy, total body areal BMD fell significantly in all groups. Only in the animals with an intact alpha estrogen receptor (WT, BERKO) could estrogens maintain bone density at baseline, preorchidectomy levels. It would appear valid to conclude that the lack of a beta estrogen receptor did not impair the effect of estrogen on the alpha estrogen receptor and that the alpha receptor alone was sufficient to prevent the loss in total body BMD caused by orchidectomy. Similarly, animals with an intact alpha estrogen receptor (WT, BERKO) were able to respond to estrogen such that femoral areal BMD and femoral bone mineral content were maintained. When femoral areal BMD and femoral bone mineral content were examined directly after orchidectomy and estrogen replacement, again only the mice with intact alpha estrogen receptors (WT and BERKO) showed levels that were higher than postorchidectomy levels. It seems that estrogen was not able to completely protect against some loss in femoral areal BMD and femoral bone mineral content. The results of these elegant experiments provide further confirmation of the importance of estrogens acting through the alpha estrogen receptors in developing and maintaining the male skeleton. The data would seem to argue that the beta estrogen receptor has little if any role to play in male skeletal metabolism. In female animals, however, age-related reductions in cancellous bone volume seem to be diminished in animals lacking the beta estrogen receptor. These observations suggest that the beta estrogen receptor may play a permissive role in age-related bone loss or that by virtue of its absence, sensitivity to the protective effects of the alpha receptor is enhanced [22,33]. Sims et al [31] provided additional evidence to support a role for the beta estrogen receptor in only female skeletal metabolism. Finally, in support of the dominant role of the alpha estrogen receptor in male skeletal physiology, the man with the alpha estrogen receptor mutation is osteoporotic [3], a telling comment on its importance in the acquisition of peak bone mass. Thus, at least in humans, the beta estrogen receptor does not have an important role to play in the acquisition of peak bone mass. Knock out of the aromatase gene has also been instructive as demonstrated by Oz et al [23] and first reported by Fisher et al [34]. The male mice

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show radiographic evidence for reduced BMD. Histologically, the mice show significant reductions in trabecular bone volume and in trabecular thickness. By histomorphometry, male knockout (but not female counterparts) show reductions in osteoblastic and osteoclastic surfaces. Female mice showed a picture more consistent with high bone turnover. This sexual dimorphism with respect to these histomorphometric features has no ready explanation at this time. Nevertheless, these animal knockout experiments provide general confirmation of the human gene knockout disorders, documenting further the important role of estrogens in male skeletal development. Studies in adult men Although the human ‘‘experiments of nature’’ initiated a major shift in our thinking regarding the role of estrogen in bone metabolism in men, they left unresolved the issue of whether estrogen primarily regulated the acquisition of peak bone mass during the growth and development of the male skeleton, or whether it also played a significant role in the maintenance of bone mass in adult men with mature skeletons. The initial attempts to address this issue came from cross-sectional observational studies in which sex steroid levels were related to BMD at various sites in cohorts of adult men. Slemenda and colleagues [35] found that BMD at various sites in 93 healthy men over age 55 years correlated with serum estradiol levels (correlation coefficients, depending on the site, of þ0.21 to þ0.35, P values 0.01 to 0.05). Somewhat surprisingly, they also found that BMD was inversely correlated with serum testosterone levels (correlation coefficients of –0.20 to –0.28, P values 0.03 to 0.10). After this report, other similar crosssectional studies have demonstrated significant positive associations between BMD and estrogen levels in men [36–42], particularly the component of circulating estradiol not bound to sex hormone binding globulin (SHBG) (ie, ‘‘bioavailable’’ estradiol). Thus, the cross-sectional observational studies show consistent positive associations between serum estrogen levels and BMD in adult men. Although these findings are compatible with the idea that estrogen plays an important role in bone metabolism in the mature male skeleton, they suffer from two potential weaknesses. First, cross-sectional analyses cannot clearly dissociate the effects of estrogen to maintain or prevent bone loss from the effects of estrogen to achieve peak bone mass. For example, a particular individual with a low bone mass at age 50 and low estradiol levels (relative to his age-matched peers) could have had life-long low estradiol levels tracking back to childhood. In this case, the low estradiol levels would reflect a deficiency in achieving peak bone mass, not necessarily an effect of estrogen to maintain or prevent bone loss. A second weakness of cross-sectional observation data is that correlation never proves causality.

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To circumvent the first of these problems, Khosla et al [43] studied in a longitudinal manner young men (22 to 39 years old) and older men (60 to 90 years old) in whom rates of change in BMD at various sites over 4 years were related to sex steroid levels. These two different age groups permitted a separate comparison of the possible effects of estrogen on the final stages of skeletal maturation versus age-related bone loss. Forearm sites (distal radius and ulna) provided the clearest data, perhaps because of the greater precision of peripheral site measurements as compared to central sites, such as the spine or hip. In the younger men, BMD at the forearm sites increased by 0.42% to 0.43% in a year, whereas in the older men, BMD at these sites declined by 0.49% to 0.66% in a year. The increase in BMD in the younger men and the decrease in BMD in the older men were more closely associated with serum bioavailable estradiol levels than with testosterone levels (Table 3). Moreover, further analysis of the data suggested that there may be a ‘‘threshold’’ bioavailable estradiol level of approximately 40 pmol/L (11 pg/ ml) below which the rate of bone loss in the older men was clearly associated with bioavailable estradiol levels, whereas above this level there did not appear to be any relationship between the rate of bone loss and bioavailable estradiol levels (Fig. 5). In these older men, a bioavailable estradiol level of 40 pmol/L (11 pg/ml) corresponded to a total estradiol level of approximately 114 pmol/L (31 pg/ml), which is close to the middle of the reported normal range for estradiol levels in men (10–50 pg/ml). These findings suggested that low estrogen levels might well contribute to the processes associated with ‘‘age-related’’ bone loss in men. This point is underscored further by the finding that bioavailable estradiol levels decline substantially with aging in men, due in large part to a marked increase in SHBG levels with increasing age [37]. Finally, these results suggest that there

Table 3 Spearman correlation coefficients relating rates of change in BMD at the radius and ulna to serum sex steroid levels among a sample of Rochester, MN men stratified by age Young

T E2 E1 Bio T Bio E2

Middle-aged

Elderly

Radius

Ulna

Radius

Ulna

Radius

Ulna

0.02 0.33** 0.35*** 0.13 0.30**

0.19 0.22* 0.34** 0.04 0.20

0.18 0.03 0.17 0.07 0.14

0.25* 0.07 0.23* 0.01 0.21*

0.13 0.21* 0.16 0.23** 0.29**

0.14 0.18* 0.14 0.27** 0.33***

* ¼ P \ 0.05. ** ¼ P \ 0.01. *** ¼ P \ 0.001. Reproduced from Khosla S, Melton III LJ, Atkinson EJ, et al. Relationship of serum sex steroid levels to longitudinal changes in bone density in young versus elderly men. J Clin Endocrinol Metab 2001;86:951–63; with permission. Abbreviations: Bio, bioavailable; E1, estrone; E2, estradiol; T, testosterone.

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Fig. 5. Rate of change in mid-radius BMD (A) and mid-ulna BMD (B) as a function of bioavailable estradiol levels in elderly men. Model R2 values were 0.20 and 0.25 for the radius and ulna, respectively, both \0.001 for comparison with a one-slope model. Solid circles correspond to subjects with bioavailable estradiol levels below 40 pmol/L (11 pg/mL) and open circles those with values above 40 pmol/L [43]. (Reproduced from Khosla S, Melton III LJ, Atkinson EJ, et al. Relationship of serum sex steroid levels to longitudinal changes in bone density in young versus elderly men. J Clin Endocrinol Metab 2001;86:951–63; with permission).

may be a difference between the reference range for estradiol in men (10– 50 pg/ml) and a physiological normal range in men ([30 pg/ml). Although this study helped to establish that estrogen levels are associated with skeletal maturation and skeletal maintenance in males, it could not definitively establish causal relationships. To address this issue, Falahati– Nini et al [44] performed a direct interventional study to distinguish between the relative contributions of estrogen versus testosterone in regulating bone resorption and formation in normal elderly men. Endogenous estrogen and testosterone production were suppressed in 59 elderly men using a combination of a long acting GnRH agonist and an aromatase inhibitor. Physiologic estrogen and testosterone levels were maintained by simultaneously placing the men on estrogen and testosterone patches delivering doses of sex steroids that mimicked circulating estradiol and testosterone levels in this age group. After baseline measurements of bone resorption (urinary deoxypyridinoline (Dpd) and N-telopeptide of type I collagen [NTx]) and bone formation (serum osteocalcin and amino-terminal propeptide of type I collagen [PINP]) markers, the subjects were randomized to one of 4 groups: Group A (T, E) discontinued the testosterone and estrogen patches; Group B (T, þE) discontinued the testosterone patch but continued the estrogen patch; Group C (þT, E) discontinued the estrogen patch but continued the estrogen patch; and Group D (þT, þE) continued both patches. Because gonadal and aromatase blockade was continued throughout the 3 week period, separate effects of estrogen versus testosterone (in the absence of aromatization to estrogen) on bone metabolism could be delineated.

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As shown in Fig. 6A, significant increases in urinary Dpd and NTx excretion, Group A (T, E), were prevented completely by continuing testosterone and estrogen replacement [Group D (þT, þE)]. Estrogen alone (Group B) was almost completely able to prevent the increase in bone resorption, whereas testosterone alone (Group C) was much less effective. Using a 2-factor ANOVA model, the effects of estrogen on urinary Dpd and NTx excretion were highly significant (P = 0.005 and 0.0002, respectively). Estrogen accounted for 70% or more of the total effect of sex steroids on bone resorption in these older men, whereas testosterone could account for no more than 30% of the effect. Using a somewhat different design, Leder et al [45] have confirmed an independent effect of testosterone on bone resorption, although the data in the aggregate clearly favor a more prominent effect of estrogen on the control of bone resorption in men. Fig. 6B shows the corresponding changes in the bone formation markers, serum osteocalcin and PINP. The reductions in osteocalcin and PINP levels with the induction of sex steroid deficiency (Group A) were prevented with

Fig. 6. Percent changes in (A) bone resorption markers (urinary deoxypyridinoline [Dpd] and N-telopeptide of type I collagen [NTx]) and (B) bone formation markers (serum osteocalcin and N-terminal extension peptide of type I collagen [PINP]) in a group of elderly men (mean age 68 years) made acutely hypogonadal and treated with an aromatase inhibitor (Group A), treated with estrogenalone (Group B), testosterone alone (Group C), or both (Group D). See text for details. Asterisks indicate significance for change from baseline: * = P \ 0.05; ** = P \ 0.01; *** = P \ 0.001. (Adapted from Falahati–Nini A, Riggs BL, Atkinson EJ, et al. Relative contributions of testosterone and estrogen in regulating bone resorption and formation in normal elderly men. J Clin Invest 2000;106:1553–60; with permission).

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continued estrogen and testosterone replacement (Group D). Serum osteocalcin, which is a marker of function of the mature osteoblast and osteocyte [46], was maintained by either estrogen or testosterone (ANOVA P values of 0.002 and 0.013, respectively). By contrast, serum PINP, which represents type I collagen synthesis throughout the various stages of osteoblast differentiation [46], was maintained by estrogen (ANOVA P value of 0.0001), but not testosterone. Collectively, these findings provided conclusive proof of an important (and indeed, dominant) role for estrogen in bone metabolism in the mature skeleton of adult men. Similar findings were subsequently reported by Taxel et al [47] in a study of 15 elderly men treated with an aromatase inhibitor for 9 weeks, where suppression of estrogen production resulted in significant increases in bone resorption markers and a suppression of bone formation markers. The dominant role played by estrogen in regulating bone resorption in men is perhaps somewhat surprising, because in vitro, estrogen and testosterone inhibit osteoclast development [48–50], enhance osteoclast apoptosis [51,52], and suppress production of the important pro-resorptive cytokine, interleukin-6 [53–56]. These findings suggest that estrogen and testosterone may regulate some other key regulator of bone resorption in opposite directions. Recently, Khosla et al [57] have reported that this may, indeed, be the case for estrogen versus testosterone regulation of osteoprotegerin (OPG), which blocks the final effector molecule for osteoclast development, receptor activator of NF-jB ligand (RANKL) [58]. In vitro [59,60] and in vivo, [57] estrogen stimulates OPG production whereas testosterone tends to inhibit it [57,61]. Although these findings do not exclude the possibility that estrogen and testosterone may have differential effects on other molecules regulating osteoclastogenesis, they do suggest that OPG may be a candidate factor accounting for the differential effects of estrogen and testosterone on bone resorption. These observations suggest a certain rationale regarding the use of estrogen in men to maintain bone mass. Because estradiol is associated with a narrow therapeutic window in terms of possible feminizing effects, a selective ER modulator (SERM) is a more attractive option. In a short-term (6 months) ‘‘proof-of-concept’’ study, Doran et al [62] treated 50 elderly men with either placebo or the SERM, raloxifene, 60 mg/d, and assessed bone turnover markers. Overall, raloxifene had no significant impact on urinary NTx excretion. The data, however, did suggest that raloxifene may reduce bone resorption in the subset of elderly men with low estradiol levels (below approximately 26 pg/ml). Clearly, further studies are needed in men with low E2 levels to test whether SERMs may have a role in preventing or treating osteoporosis in men. In summary, the evidence is now overwhelming that estrogen plays a critical role in regulating bone metabolism in men. What began as surprising and novel observations in ER-a and aromatase deficient males

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has now been supported by a large body of data in animals and in humans clearly demonstrating the estrogen regulates bone resorption and bone formation in males, with important effects on BMD in cross-sectional and in longitudinal studies.

Role of androgens in bone metabolism in women Human ‘‘experiments of nature’’ Although there is no precise female counterpart to the estrogen receptor alpha deficient male (ie, females with complete androgen insensitivity), evidence from the testicular feminization males, who have inactivating mutations in the androgen receptor gene, is compatible with an important role for androgens in the (phenotypic) female skeleton. In these patients, failure to respond to androgens does not delay epiphyseal closure, but several case reports and small series [63–65] have suggested that these patients do have low BMD at several sites. Perhaps the most comprehensive assessment of BMD in these individuals was published by Marcus et al [66] who studied 28 patients with either complete (22 subjects) or partial (6 subjects) androgen insensitivity. Because many of these patients undergo prophylactic orchidectomy to prevent the occurrence of testicular cancer, the investigators also attempted to control for compliance with estrogen replacement, because that is often a confounding factor in studies with these patients. BMD was reduced in these patients, including in those subjects complying with estrogen replacement therapy, consistent with an important (albeit not dominant, as in the case of estrogen in the estrogen receptor alpha and aromatase deficient males) role for androgens in bone metabolism in these phenotypic females. Moreover, the deficits in BMD tended to be more severe in the subjects with complete, as compared to partial androgen insensitivity. Studies in experimental animals The animal counterpart to the testicular feminization patients, the testicular feminized rat, has also been characterized for its skeletal phenotype. These animals have reductions in bone size (femoral length, diameter, and cortical thickness) compared to wild-type male animals [67]. However, they have a relative preservation of cancellous BMD at various sites [67]. Thus, as has been the case for the human estrogen receptor alpha deficient male compared to their animal counterparts, the phenotypic consequences of deletion of the AR are not necessarily the same in humans versus rodents. The testicular feminization rat does, however, highlight what is perhaps the major effect of androgens on the skeleton—the regulation of bone size, most likely by effects on enhancing the apposition of bone on the outer, periosteal surface. This effect likely accounts, at least in part, for the sexual

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Fig. 7. The effect of ovariectomy (A) and orchidectomy (B) on periosteal bone formation rate. Intact controls (open circles and triangles), ovariectomized and orchidectomized animals, respectively ( filled circles and triangles). P \ 0.01 for all ovariectomy and orchidectomy time points compared to intact controls. (Adapted from Turner RT, Wakley GK, Hannon KS. Differential effects on androgens on cortical bone histomorphometry in gonadectomized male and female rats. J Orthop Res 1990;8:612–7; with permission).

dimorphism of the skeleton that develops at the time of puberty in humans and most mammalian species. The specific role of androgens in mediating this was perhaps best demonstrated by Turner and colleagues [68], who found that ovariectomy in female rats resulted in an increase in periosteal bone formation rate, whereas orchidectomy in male rats resulted in a decrease in this parameter (Fig. 7). Several studies in rats have also documented the efficacy of the nonaromatizable androgen, 5a-dihydrotestosterone, in at least partially restoring bone mass following ovariectomy [69–72]. These studies have generally found predominant effects of 5a-dihydrotestosterone on bone formation indices, with high doses being required in one study to demonstrate effects on reducing bone resorption parameters [71]. Thus, consistent with the findings in humans, androgens (whereas they likely have antiresorptive effects) appear to be less potent than estrogen in inhibiting bone resorption, and their major beneficial effect may be on stimulating bone formation. The potential regulatory role of adrenal androgens in bone metabolism has also been assessed in several animal studies. Durbridge et al [73] found that, in female rats, adrenalectomy alone resulted in loss of metaphyseal trabecular bone of an extent similar to that produced by ovariectomy. In addition, Turner et al [74] showed that treatment with the adrenal androgen, DHEA, reduced the loss of cancellous bone following ovariectomy in female rats, indicating that adrenal androgens may prevent the bone loss induced by estrogen deficiency. Finally, Lea et al [75] showed not only that the adrenal androgen, androstenedione, could reduce loss of cancellous bone volume in ovariectomized rats but that this effect was not prevented by simultaneous

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treatment with an aromatase inhibitor, implying that the effect of androstenedione was not mediated by way of conversion to E. Studies in adult women At the other end of the spectrum to the testicular feminization patients are women with hyperandrogenic states, such as polycystic ovary syndrome (PCOS). These women generally have increases in BMD [76], although because they also tend to be obese, it is often difficult to separate the effects of obesity on bone mass from those of increased androgen levels. In a study specifically examining lean women with PCOS compared to weight matched controls, BMD was similar in the two groups at most sites, except for the arms and ribs [77]. Thus, although women with hyperandrogenic states may well have increased BMD, the evidence is far from unequivocal at this point. In addition, it is also possible that the ultimate effect of the excess androgens on bone in these women is, in fact, by way of local aromatization of androgens to estrogens. Several observational studies in normal women have also found associations between serum androgen levels and BMD. In premenopausal women, total and bioavailable testosterone levels are significantly associated with BMD at the proximal femur [37]. Moreover, serum testosterone levels appear to be the most robust predictors of bone loss at the femoral neck in these women, even after adjusting for important covariates, such as age, weight, and serum estradiol levels [78]. Data on associations in women between BMD and adrenal androgen levels, such as DHEA and its sulfated ester, DHEAS, are somewhat conflicting. Thus, some [79,80], but not all [81,82], studies have found positive associations between serum DHEAS levels and BMD at various sites. In addition, postmenopausal women with primary adrenal failure have been found to have decreased circulating DHEA levels and deficits in distal forearm BMD as compared to control postmenopausal women [83], suggesting a role for adrenal androgens in the maintenance of bone mass. Several studies have used androgens (either low doses of testosterone, adrenal androgens, or synthetic androgens), primarily in combination with E, to attempt to increase BMD in postmenopausal women. The rationale for these studies has generally been that because estrogen is known to have potent anti-resorptive effects on bone, the addition of an androgen may result in enhanced efficacy through effects of the latter on stimulating bone formation. Consistent with this, Raisz et al [84] found that postmenopausal women treated with 1.25 mg of oral esterified estrogen plus 2.5 mg of methyltestosterone daily had a 24% increase in the bone formation marker, serum osteocalcin, after 3 months of treatment, compared to 40% lower values in women treated with estrogen alone. Similar findings were noted for the other bone formation markers, bone-specific alkaline phosphatase and C-terminal procollagen peptide. In a longer term study, Watts et al [85]

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compared the effects of 2 years of treatment of surgically menopausal women with either 1.25 mg of oral esterified estrogens or estrogen plus 2.5 mg daily of methyltestosterone. They found that the combination treatment resulted in a significant, 3.4% increase in spine BMD, whereas estrogen alone resulted only in maintenance of BMD. However, the difference between the two groups was not statistically significant. Data on possible effects of adrenal androgens on BMD in postmenopausal women are sparse at present. In an uncontrolled study, Labrie et al [86] found that daily application of 10% DHEA cream in 14 postmenopausal women resulted in a significant (2%) increase in hip BMD associated with a decrease in bone turnover markers. Finally, there have been several studies using synthetic androgens, such as methandrostenole [87] or nandrolone decanoate [88–92], showing beneficial effects on BMD in postmenopausal women, although the virilizing and adverse metabolic side-effects of these agents has generally dampened enthusiasm for their widespread clinical use. In summary, as is the case for estrogen in men, androgens do appear to impact bone metabolism in women. Whereas estrogen seems to play a critical role in the acquisition and maintenance of bone mass in men, the major effects of androgens in women (and in men) may be on increasing bone size and enhancing bone formation, with some effects on inhibiting bone resorption. Thus, although androgens are likely important for bone metabolism in women, they do not appear to play the central role that estrogen plays in regulating skeletal function in both genders. Molecular underpinnings of the gender non-specificity of sex steroid action The effects of estrogen on bone metabolism in males and conversely, of androgens in females may, in large part, be mediated by actions of estrogen and androgens by way of their cognate receptors. On the other hand, there has recently been intriguing and provocative data indicating that estrogen and testosterone may be able to mediate at least some of their effects not only by way of their own receptors, but also through actions of estrogen on the androgen receptor and testosterone (or testosterone metabolites) on the estrogen receptor. Although it is clear that the traditional, transcriptional effects of estrogen and testosterone are likely mediated by way of the estrogen and androgen receptor, respectively, Kousteni et al [7] have found that the effects of sex steroids on preventing osteoblast apoptosis, which are mediated by way of non-genomic actions involving activation of a Src/Shc/ERK signaling pathway, appear to be gender-nonspecific. These effects are mediated by the ligand (rather than DNA) binding domain of estrogen receptor alpha, estrogen receptor beta, or androgen receptor, and can be transmitted with similar efficiency irrespective of whether the ligand is estrogen or an androgen. Moreover, the estrogen receptor antagonist, ICI 182,780 can block either estrogen or testosterone actions on preventing apoptosis, and the androgen

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Fig. 8. Anti-apoptotic effects of estrogen and 5a-dihydrotestosterone (DHT) on osteoblastic cells and evidence for sex-nonspecific signaling. Mouse calvarial osteoblasts were pretreated with the estrogen receptor antagonist ICI 182,780 (107 M) or with the androgen receptor antagonist flutamide (107 M) for 30 minutes, followed by incubation with 108 M estradiol or DHT for 1 hour. Subsequently, the apoptosis inducer, etoposide, was added and apoptotic cells were quantified after 6 hours. * = P \ 0.05 versus vehicle, by ANOVA. (Adapted from Kousteni S, Bellido T, Plotkin LI, et al. Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 2001;104:1–20; with permission).

receptor antagonist, hydroxyfultamide, can also block estrogen and testosterone effects (Fig. 8). Further evidence for this molecular cross-talk comes from studies in the mouse prostate demonstrating that the testosterone metabolite, 5aandrostane-3b,17b-diol, can bind estrogen receptor beta and thereby reduce prostatic androgen receptor levels [8]. This, in turn, results in reduced proliferation of the epithelial cells of the prostate. Whether androgen metabolites have similar effects by way of estrogen receptor beta in other tissues, such as bone, is at present unclear, but certainly an intriguing possibility. Summary The past years several have witnessed a significant transformation in our understanding of sex steroid action in the male and female skeleton. Data from animal and human studies indicate that sex steroids have important skeletal effects in both genders. It seems from the in vivo human data that estrogen is likely more potent than testosterone in inhibiting bone resorption. Estrogen and testosterone appear to be important for maintaining bone formation. In addition, androgens clearly enhance bone size, likely through effects on periosteal bone formation. How much of this gender crosstalk at the physiological level is caused by ‘‘promiscuous’’ actions of sex steroids at the molecular level, with estrogen acting by way of the androgen receptor (and androgens via the estrogen receptor) is an interesting and

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