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Laboratory for Experimental Medicine and Endocrinology (D.V., L.V., S.B., R.B.), and Leuven University Centre for Metabolic Bone Diseases and Division of Geriatric Medicine (S.B.), Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; and Division of Endocrinology, Department of Internal Medicine (M.K.L., C.O.), Sahlgrenska University Hospital, SE-41345 Göteborg, Sweden
Correspondence: Address all correspondence and requests for reprints to: C. Ohlsson, M.D., Ph.D., Division of Endocrinology, Department of Internal Medicine, Sahlgrenska University Hospital, SE-41345 Göteborg, Sweden. E-mail: Claes.Ohlsson{at}medic.gu.se
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Abstract |
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 Top Abstract I. IntroductionII. General Aspects of...III. Effects of Androgens...IV. Effects of Androgens...V. Indirect Mechanisms of...VI. Effects of Androgens...VII. General ConclusionsReferences |
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Androgens also increase cortical bone size via stimulation of both longitudinal and radial growth. First, androgens, like estrogens, have a biphasic effect on endochondral bone formation: at the start of puberty, sex steroids stimulate endochondral bone formation, whereas they induce epiphyseal closure at the end of puberty. Androgen action on the growth plate is, however, clearly mediated via aromatization in estrogens and interaction with ER
. Androgens increase radial growth, whereas estrogens decrease periosteal bone formation. This effect of androgens may be important because bone strength in males seems to be determined by relatively higher periosteal bone formation and, therefore, greater bone dimensions, relative to muscle mass at older age. Experiments in mice again suggest that both the AR and ER pathways are involved in androgen action on radial bone growth. ERß may mediate growth-limiting effects of estrogens in the female but does not seem to be involved in the regulation of bone size in males.
In conclusion, androgens may protect men against osteoporosis via maintenance of cancellous bone mass and expansion of cortical bone. Such androgen action on bone is mediated by the AR and ER.
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I. Introduction |
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TopAbstractI. IntroductionII. General Aspects of...III. Effects of Androgens...IV. Effects of Androgens...V. Indirect Mechanisms of...VI. Effects of Androgens...VII. General ConclusionsReferences |
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In the early 1940s, Albright and Reifenstein were among the first to refer to the antiosteoporotic and anabolic properties of androgens (2). From a public health perspective, osteoporosis is a greater problem in women than in men (3). This explains why most research efforts to explore skeletal effects of sex steroids have been devoted to estrogens. Moreover, androgens may be converted into estrogens via the P450 aromatase enzyme complex and may therefore act as prohormones for estrogens. In this respect, there is increasing evidence that at least part of the effects of androgens in men can be explained by their aromatization into estrogens (4, 5). Epiphyseal closure at the end of puberty, for example, is now generally accepted to be estrogen-dependent in both genders (6). In recent years, a specific role of androgens in skeletal homeostasis has even been questioned, although androgen receptors (ARs) in bone cells and AR-mediated actions on bone have been documented for more than a decade (7, 8).
The aim of this review is to address the question whether and how (through which receptors and/or pathways) androgens may affect bone strength and provide protection against osteoporosis. The clinical relevance of this question results from the recognition that, even in men, fractures due to skeletal fragility represent a huge public health problem.
Elderly men maintain cancellous bone integrity in comparison with postmenopausal women, although their bone trabeculae become thinner. From a biomechanical perspective, compromised bone strength in men is the result of gender-related differences in the loss of bone mass during aging and, even more importantly, in the accumulation of bone mass during childhood and adolescence (9) (Table 1
). During puberty, men develop a bigger bone size than women, due to increased periosteal apposition. In females, on the other hand, estrogens have an inhibitory effect on periosteal bone formation, whereas endocortical apposition is stimulated, narrowing the medullary cavity. Estrogens also stimulate epiphyseal closure earlier in women, resulting in longer bones in men. After puberty, the amount of bone formed on the periosteal surface is still greater in men (10, 11), whereas endocortical bone resorption is similar in both sexes, so that net bone loss is less in men (Table 1). The end result is a skeletal sexual dimorphism, characterized by a greater bone length, larger outer and inner bone perimeters, and a larger cortical volume in men compared with women. Therefore, adult men have greater bone mass than women, but this is due to a greater bone volume and not to a greater volumetric density. The greater areal bone mineral density (BMD) in males is thus only an artifact of the dual energy x-ray absorptiometry (DXA) software by expression of bone mass as projected areal (grams per square centimeter) instead of true or volumetric density (grams per cubic centimeter). In the current review, we will focus on the potential mechanisms through which androgens may prevent bone loss, increase bone mass (size), and improve bone strength.
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Finally, the potential clinical benefits of androgen replacement in the context of male hypogonadism as well as in different patient groups will be discussed. Recent studies have explored different modes of action of androgens via the AR and ER pathways and may ultimately contribute to the potential use of selective AR or ER modulators in selected male target populations.
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II. General Aspects of Androgen Action |
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TopAbstractI. IntroductionII. General Aspects of...III. Effects of Androgens...IV. Effects of Androgens...V. Indirect Mechanisms of...VI. Effects of Androgens...VII. General ConclusionsReferences |
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-reductase to the more potent 5-dihydrotestosterone (DHT). Both DHT and T can activate the AR (Fig. 1). T can also be converted to estradiol (E2) by an enzyme complex known as estrogen synthetase or aromatase followed by activation of the ERs. The adrenal cortex secretes large amounts of C-19 androgens including dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEA-S), and androstenedione. These C-19 androgens can be metabolized either directly or indirectly in a rather complex pathway to estrone by the aromatase enzyme or to T by steroid sulfatase, 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and/or 3ß-HSD (Fig. 1). Thus, depending on the relative activity of aromatase, 5-reductase, 17ß-HSD, 3ß-HSD, and steroid sulfatase (13), T and C-19 androgens may predominantly activate either the AR or the ERs (Fig. 1). Several recent publications have demonstrated that aromatase (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25), 5-reductase (14, 17, 18, 22, 26, 27, 28, 29, 30), 17ß-HSD (14, 15, 16, 17, 19, 23, 27, 31), 3ß-HSD (14, 32), and steroid sulfatase (13, 14, 15, 17, 19) are expressed in bone tissue, supporting the notion that local metabolism of androgens in bone tissue might be of physiological importance. Thus, sex steroids are partly synthesized locally in peripheral tissues, providing individual target tissues with the means to adjust formation and metabolism of sex steroids to their local requirements (Fig. 1). To elucidate the relative importance of androgen metabolism and action in bone, a variety of specific inhibitors/antagonists have been extensively used, including 5-reductase inhibition (33, 34, 35), AR antagonists (36, 37, 38, 39, 40), AR inactivation in rats (41) and mice (42, 43, 44, 45, 46), aromatase inhibitors (47, 48, 49), aromatase gene inactivation [in humans (50, 51, 52, 53) and mice (54, 55, 56)], ER antagonists (36, 40, 57, 58, 59, 60), ER gene inactivation [in humans (61) and mice (46, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74)], and ERß inactivation in mice (64, 65, 66, 67, 73, 75, 76, 77, 78, 79, 80, 81) (Fig. 1). A large part of this review will describe and discuss the results of these different approaches to improving our understanding of the mechanism of action of androgens in bone tissue.
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, and ERß in mediating the effect of androgens on bone. The AR was cloned in 1988 (82, 83). ER was cloned in 1986 (84, 85) and a second ER, ERß, in 1995 (86). All of these receptors belong to the nuclear receptor family. They are all composed of three independent but interacting functional domains; the NH2-terminal or A/B domain, the C or DNA-binding domain, and the D/E/F or ligand-binding domain (LBD) (87) (Fig. 2). The sex steroid receptors are DNA-binding proteins that have the capacity to interact with specific DNA sequences, the androgen response element for the AR, and the estrogen response element for the ERs. The sequence homology in the DNA-binding domain is high among the sex steroid receptors (87). The N-terminal domain of these receptors encodes a ligand-independent activation function (AF-1), a region of the receptor involved in protein-protein interactions, and transcriptional activation of target gene expression (87) (Fig. 2). The COOH-terminal region, or LBD, mediates ligand binding, receptor dimerization, nuclear translocation, and transcription of target gene expression (87) (Fig. 2). The classical mechanism of sex steroid action involves interaction with intracellular receptors, which are either cytoplasmic or nuclear. Binding of the sex steroids to their respective receptors leads to conformational changes of the protein that allow it to interact with the transcriptional machinery: directly or indirectly via protein-protein interactions with different transcription factors (88). The transcriptional activity of androgen-bound AR and estrogen-bound ERs is affected by tissue-specific coregulators, including factors enhancing transactivation (coactivators) and factors reducing transactivation (corepressors) (87, 89).
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B proteins (87). Furthermore, a variety of cell types respond to estrogens rapidly (within seconds/minutes), making a classical genomic mechanism of action unlikely (90). The importance of nongenomic mechanisms, in which the ligand interacts with plasma membrane/cytosolic receptors, is increasingly recognized to mediate the rapid responses to sex steroids (8, 91, 92). Nongenomic rapid effects of estrogens in vitro have been described for both osteoblasts and osteoclasts (8, 88, 91, 93). Furthermore, a plasma membrane ER is reported to partly mediate a nongenomic apoptotic effect of estrogens on osteoclasts (94).
The group of Manolagas (88) has demonstrated that the antiapoptotic effect of estrogens and androgens on osteoblasts in vitro is mediated by Src/Shc/ERK signaling via a nongenomic action of the classical receptors and is sex nonspecific. This action is mediated by the LBD and is eliminated by nuclear targeting of the receptor protein (Fig. 3A). More recently, the same research group presented in vitro as well as in vivo data suggesting that the nongenomic effect of sex steroids involves kinase-mediated regulation of common transcription factors (95). Thus, nongenomic effects of sex steroids alter the activity of Elk-1, CCAAT enhancer binding protein-ß (C/EBPß), and cAMP-response element binding protein, or c-Jun/c-Fos by an extranuclear action of the ER or AR, resulting in activation of the Src/Shc/ERK pathway or down-regulation of c-Jun N-terminal kinase, respectively (95). Interestingly, a synthetic ligand, estren, which reproduces the nongenomic effects of sex steroids without affecting classical transcription, increases BMD in both ovariectomized (ovx) females and orchidectomized (orch) male mice, without affecting reproductive organs (uterus and seminal vesicles) (Fig. 3B). Such ligands merit investigation as potential therapeutic alternatives to hormone replacement for osteoporosis, in both women and men (88, 95, 96).
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, ERß, and the AR can transmit the antiapoptotic effect of sex steroids with similar efficiency, irrespective of whether the ligand is an estrogen or an androgen. In contrast, however, several in vivo studies of transgenic mouse models do not support the notion that estrogens have important AR-mediated physiological effects on cancellous BMD or the concept that nonaromatizable androgens exert bone-sparing effects on cancellous BMD through direct activation of the ERs (42, 45, 66, 67, 70, 98) (see also Sections IV.Eand IV.F). Although the hypothesis by Manolagas et al. that the bone-sparing effect of sex steroids is mediated via nongenomic mechanisms is extremely interesting and provocative, additional investigation and confirmation by others are required before it can be fully accepted (99).
D. Expression of androgen and estrogen receptors in the skeleton
It is generally believed that an important part of the effect of androgens or their metabolites on the skeleton is exerted via a direct stimulation of the AR, ER, and/or ERß expressed locally in the skeleton. In this section, we will summarize in vitro and in vivo studies investigating the expression of these three receptors by growth plate chondrocytes, osteoblasts, osteocytes, osteoclasts, and/or by other bone-related cells.
1. Growth plate cartilage.
Androgens exert important effects on pubertal growth, and a local effect on the growth plate is supported by the fact that both cultured epiphyseal chondrocytes (100) and growth plate cartilage cells in vivo express AR (101, 102, 103, 104, 105) as detected by immunohistochemistry (101, 102, 103, 104, 105), binding studies (100), and in situ hybridization (105) (Table 2). The AR has been detected in all layers of the human growth plate at different ages (101, 102, 103, 104), whereas in the rat it was expressed in proliferative and early hypertrophic chondrocytes at sexual maturation and only in prehypertrophic chondrocytes in older rats (105). Male rats displayed a higher AR expression in the growth plate and metaphyseal bone than female rats during sexual maturation (105). In contrast, no major sex difference regarding AR expression has been observed in human growth plate chondrocytes (100, 101, 102).
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(102, 104, 106, 107, 108, 109, 110, 111, 112) and ERß (104, 106, 110, 113) protein in the human, rabbit, and rat growth plate by using immunohistochemistry (Table 2
), and the results regarding ER have been confirmed by in situ hybridization (107). Most studies have detected ER expression in all layers of the human and rabbit growth plate during both fetal stage and puberty (102, 104, 108, 110). In contrast, Kennedy et al. (111) detected ER expression in the growth plate of immature but not mature rats. In addition, it is clear that the ERß protein is expressed in the growth plate, but the expression pattern varies between studies. The first study by Nilsson et al. (113) localized ERß mainly to the hypertrophic chondrocytes, whereas later studies have detected it in all layers of the growth plate (104, 106, 110) (Table 2). In conclusion, AR, ER, and ERß are all expressed in the growth plate, indicating that androgens, either directly or after aromatization, might influence the pubertal growth spurt and growth plate closure via a direct interaction with local sex steroid receptors. Future experiments, using growth plate-specific inactivation of the different sex steroid receptors, are needed to investigate whether these locally expressed receptors are of functional importance for the regulation of longitudinal bone growth.
2. Osteoblasts/osteocytes.
Despite the obvious importance of androgens and estrogens in the regulation of adult bone metabolism, it has been difficult to detect AR and ERs in osteoblasts. Therefore, osteoblasts were for a long time not generally considered as primary target cells for sex steroids. The development of new and more sensitive techniques has resulted in the detection of the AR as well as ER and ERß expression by osteoblasts and osteocytes. The expression of the AR in cultured osteoblasts was first described in 1989 by Colvard et al. (114) using a nuclear binding assay. Several in vivo and in vitro studies have confirmed that the AR mRNA and protein are expressed by osteoblasts and osteocytes (14, 18, 101, 103, 105, 114, 115, 116, 117, 118, 119, 120, 121, 122) (Table 3). AR binding has been demonstrated in vitro in osteosarcoma cell lines, osteoblast-like cell lines, and primary osteoblasts from several different species including human, rat, and mouse (18, 115, 118, 119, 120, 123, 124, 125, 126) (Table 3). The number of binding sites per cell appears to vary greatly from 70 to 14,000 binding sites per cell (127), depending on the assay technique, but it is in a range seen in other androgen target tissues. Human osteoblastic cells, isolated from cortical bone, expressed higher AR mRNA levels and AR binding than cells isolated from cancellous bone, whereas no major differences in AR expression in osteoblasts derived from males compared with osteoblasts derived from females were found (118). Most studies (116, 117, 119, 125), but not all (118, 128), indicate that androgen up-regulates the expression of its own receptor in osteoblasts.
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). The number of estrogen binding sites in different studies varies between 60 and 4,500 binding sites per osteoblast, which is lower than in estrogen-responsive reproductive cells such as uterine and breast cells (132). Estrogens regulate osteoblast proliferation and expression of genes encoding enzymes, bone matrix proteins, hormone receptors, transcription factors, as well as growth factors and cytokines (133). However, conflicting results have been presented regarding the in vitro effects of estrogens on proliferation and differentiation of osteoblasts, which might be due to differences in the number of receptors per cell, animal species, skeletal localization of the osteoblasts, the stage of osteoblast differentiation, and the relative concentration of ER vs. ERß in the osteoblasts (132, 133). It is now well-established that both ER and ERß are expressed by osteoblasts and osteocytes, as studied both in vivo and in vitro; in several different species including human, rat, mouse, pig, and guinea pig; and using several different techniques including Western blot, Northern blot, RNase protection assay, PCR, in situ hybridization, and immunohistochemistry (Table 3). Some studies indicate that ER expression increases with the increasing stage of differentiation of cultured osteoblasts (132, 134, 135, 136). ERß mRNA levels have been shown to either increase (135) or remain constant (134) with advancing cellular development. Thus, the ratio of ER to ERß and the estrogenic response may vary as the cells progress from preosteoblasts to mature osteoblasts (132). In a recent paper, the relative expression of ER, ERß, and the AR was followed simultaneously during differentiation of cultured osteoblasts (121), demonstrating that ER levels were elevated during matrix maturation and then declined during mineralization. ERß expression was relatively constant throughout differentiation, whereas AR levels were lowest during proliferation and then increased throughout differentiation, with highest levels in the most mature mineralizing cultures (121). One study in human developing bone demonstrated that ER immunoreactivity is strong in osteoblasts adjacent to the periosteal surface of the cortical bone, whereas ERß immunoreactivity is dominant in osteoblasts in cancellous bone (106). Taken together, the AR and both ER and ERß are expressed by osteoblasts, but there is no consensus about their relative expression during differentiation and their localization within the skeleton.
3. Osteoclasts.
AR expression has been detected in avian (137) and mouse (138) osteoclasts in vitro and in rat osteoclasts in vivo (17), whereas no expression has been detected in human osteoclasts in vivo (101, 103) (Table 4). Thus, the available data are contradictory regarding AR expression in osteoclasts, and it is generally believed that the major part of the effect of androgens on osteoclastogenesis and bone resorption is mediated by cells of the osteoblast lineage (139). However, some recent in vitro studies demonstrate that androgens can act directly on osteoclasts to promote their apoptosis (88, 96, 140). Furthermore, in bone marrow cell preparations, sex steroids have identical effects on osteoclastogenesis in the presence or absence of cells of the osteoblastic lineage (140). An effect of estrogens, produced by aromatization of androgens, via ERs localized in osteoclasts is possible because most (106, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151), but not all (107, 152, 153), studies have identified ER and ERß in osteoclasts (Table 4). In two separate studies, preosteoclasts, but not mature osteoclasts, were found to express ER (154, 155). Taken together, the conflicting results regarding ER expression in osteoclasts suggest that osteoclasts express a low number of ERs, which may be close to the detection limit of the different assays used. This would be consistent with the fact that most but not all studies have detected ERs on osteoclasts. Although the expression and the physiological role of sex steroid receptors in osteoclasts remain controversial, the available evidence suggests that the inhibitory effect of estrogens on osteoclastogenesis is largely mediated indirectly by cells of the osteoblast lineage, and not via a direct interaction with ERs on osteoclasts.
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(122, 154, 157, 158, 159, 160) and ERß (122, 157, 158) (Table 5). Furthermore, both AR and ERs have been detected on megakaryocytes (151, 156) and endothelial cells (101, 151, 156) within the bone compartment. Thus, besides growth plate chondrocytes and osteoblasts, it is clear that several other types of cells within the skeleton express sex steroid receptors, which may be involved in mediating the effect of androgens on the skeleton.
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III. Effects of Androgens in Vitro on Skeletal Cells |
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TopAbstractI. IntroductionII. General Aspects of...III. Effects of Androgens...IV. Effects of Androgens...V. Indirect Mechanisms of...VI. Effects of Androgens...VII. General ConclusionsReferences |
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A. Growth plate chondrocytes
Androgens probably have direct effects on growth plate cartilage and thus on longitudinal bone growth. It has been documented, at least when using very strict culture conditions, that androgens regulate both proliferation and differentiation of cultured epiphyseal chondrocytes (100, 161, 162, 163, 164), supporting a direct effect of androgens on growth plate cartilage. A direct effect of androgens on epiphyseal growth and maturation is also supported by the fact that T, injected directly into the growth plate of rats, increases the growth plate width (165). Androgens, however, also have an important effect on GH secretion and its pulsatility during puberty, and this may indirectly mediate their effects on linear growth (166).
B. Osteoblasts/osteocytes
Most in vitro studies (18, 126, 167, 168, 169, 170, 171, 172), but not all (115, 173, 174), demonstrate that both DHT and T increase cell proliferation of cultured osteoblast progenitors derived from different species. The effects on osteoblast differentiation are rather controversial, including stimulatory, no effect, and inhibitory effect on alkaline phosphatase, type I collagen, osteocalcin, and mineralization of extracellular bone matrix (18, 115, 125, 167, 169, 170, 173, 174, 175, 176, 177). These conflicting results might be due to differences in receptor concentration, animal species, skeletal localization of the osteoblasts, or the stage of osteoblast differentiation. However, in our opinion, most studies indicate that androgens induce a more differentiated osteoblast phenotype. Recent studies demonstrate that androgens decrease osteoblast and osteocyte apoptosis (88, 96). Thus, most in vitro studies support the notion that androgens stimulate proliferation of osteoblast progenitors and differentiation of mature osteoblasts while inhibiting apoptosis of osteoblasts.
Some of the local effects of androgens on bone might, as previously described for estrogens (4), be mediated via a regulation of cytokines and growth factors expressed locally in bone. The three most discussed androgen-regulated locally expressed factors include TGFß, IGFs, and IL-6. TGFß and the IGFs are involved in bone formation, whereas IL-6 increases osteoclastogenesis and androgens may therefore theoretically preserve bone via either an induction of TGFß and IGFs or an inhibition of IL-6. We will here discuss results regarding androgen-induced regulation of these factors, but it should be emphasized that the functional role of these regulations is complex and full interpretation is not yet possible. TGFß is highly expressed in bone tissue (the largest reservoir for TGFß), and it is a mitogen for osteoblasts (178, 179). Several studies, both in vivo and in vitro, indicate that androgens increase TGFß expression and/or activity (115, 169, 180, 181). Furthermore, orch reduces bone content of TGFß, whereas T treatment increases TGFß content (181). It remains unclear to what extent this effect of T is mediated through the AR or ERs. In contrast, Hofbauer et al. (173) found that androgens decrease TGFß mRNA levels in a human osteoblastic cell line. IGFs and IGF-binding proteins (IGFBPs) exert important effects on osteoblast proliferation and differentiation (182, 183). Androgens have been shown to regulate the expression and/or the activity of IGFs either directly by regulating IGF expression or indirectly via a regulation of the expression of IGFBPs in several (168, 175, 184), but not all, studies (18, 185). IL-6 is a cytokine believed to be involved in the bone loss associated with sex steroid deficiency. It increases osteoclastogenesis and bone resorption. Orch increases IL-6 secretion by bone marrow cells (186). DHT and T suppress the IL-6 production in both cultured bone marrow stromal cells and osteoblasts (187, 188, 189). Furthermore, androgens inhibit the expression of the gp80 and the gp130 subunits of the IL-6 receptor (190). Orch increases osteoclastogenesis, which is inhibited by androgens or IL-6 neutralizing antibody (188), and IL-6 knockout mice do not lose bone after orch (188), supporting the notion that inhibition of IL-6 production is at least partly involved in the antiresorptive effect of androgens on bone. Thus, TGFß, IGFs, and IL-6 are three major factors believed to be involved in the bone-sparing effect of androgens. However, other possible pathways for androgen regulation of bone metabolism have also been investigated. Androgens inhibit PTH- or IL-1-induced prostaglandin E2 production (176) and PTH-induced cAMP production (170, 191), whereas they increase IL-1ß production (192) and the mitogenic effect of fibroblast growth factor (168) in cultured osteoblasts. A recently published study demonstrates that DHT decreases osteoprotegerin (OPG) levels (193), whereas it has previously been shown that estrogens increased OPG expression in cultured osteoblasts (194, 195). It remains to be investigated in vivo whether DHT and estrogens have opposite effects on OPG expression and, if so, whether it is of any physiological importance for the regulation of bone homeostasis.
C. Osteoclasts
Osteoclasts are derived from hematopoietic precursor cells of the colony-forming unit granulocyte-macrophage lineage within the bone marrow. The proliferation of these colony-forming unit granulocyte macrophages is up-regulated after orch. The terminal differentiation into mature osteoclasts requires close interaction and also cell-to-cell contact with stromal cells of the osteoblastic lineage in the bone marrow under tight control of the receptor activator of nuclear factor B-ligand (RANKL)/OPG system (196). The osteoblastic stromal cells also appear to be essential for the bone-sparing action of androgens. This notion is supported by the fact that in the SAMP6 mouse model, in which osteoblast function is impaired due to an age-related decrease in osteoblast progenitors (197), the rise in remodeling after orch is blunted. Their failure to up-regulate osteoclastogenesis is secondary to defective osteoblast formation. Thus, orch-induced increased osteoclastogenesis is dependent on osteoblast/preosteoblast-derived signals (139). Therefore, the removal of testes-derived sex steroids by orch in mice primarily leads to bone marrow changes. These changes, mediated by cells of the osteoblast lineage, are characterized by an increase of osteoblast precursors, which in turn indirectly stimulates osteoclastogenesis (198). A direct effect of androgens on preosteoclasts/osteoclasts is more controversial. However, Pederson et al. (137) have demonstrated that osteoclasts express AR and that DHT inhibits the resorptive capacity of isolated human, murine, and avian osteoclasts in vitro. Furthermore, recent in vitro data suggest that androgens may also directly modulate RANKL-induced osteoclast formation, independently of the bone marrow cells (199). Additionally, androgens, like estrogens, may regulate osteoclast survival, RANK expression in preosteoclasts, and activity of mature osteoclast independently of their effects on bone marrow stromal cells, at least in vitro. However, the contribution of these in vitro observations to in vivo activity of androgens remains to be clarified. A direct effect of estrogens on osteoclasts in vivo is supported by the finding that E2 promotes apoptosis of murine osteoclasts (200). These results indicate that osteoclast precursors as well as osteoclasts are able to respond directly to androgens in vitro and thus are potential androgen target cells in vivo (137). In conclusion, it is apparent that some of the effect of androgens on osteoclastogenesis is indirectly mediated via cells of the osteoblasts lineage, although further investigation is needed to characterize a possible direct effect of androgens on osteoclasts in vivo.
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IV. Effects of Androgens on the Rodent Skeleton |
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TopAbstractI. IntroductionII. General Aspects of...III. Effects of Androgens...IV. Effects of Androgens...V. Indirect Mechanisms of...VI. Effects of Androgens...VII. General ConclusionsReferences |
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Young rats have been widely used as a model for the growing skeleton. The skeleton of rats, though, differs from the human skeleton because the growth plates never fully close (201). This should not be overinterpreted because by 12 months of age, the growth plate characteristics have stabilized, with no further significant change up to 24 months (202). This allows aged rats to be used as a model for human skeletal remodeling. Bone remodeling maintains the mechanical and structural integrity of the skeleton after puberty. Coupling of osteoblast and osteoclast actions ensures that the processes of bone resorption and formation occur at the same time and place, which allows old bone to be replaced by new bone. It is also important to mention that rodents do not experience spontaneous fractures. Rodent studies will therefore not answer the question whether androgens protect against osteoporotic fractures, but they may still contribute to our knowledge of how androgens influence skeletal structure and density.
Several experimental procedures have been used to evaluate skeletal androgen action and metabolism in male and female rodents. These include (surgical and chemical) castration and administration of AR antagonists, ER antagonists, aromatase inhibitors, selective ER modulators (SERMs), and type II 5-reductase inhibitors, either alone or in combination with sex steroid replacement. Because the skeletal effects of these experimental conditions often differ between cortical and cancellous bone compartments, these compartments will be considered separately. Some of these interventions may also induce extraskeletal effects—including changes in body composition, growth, and food intake—that may indirectly interfere with skeletal homeostasis (see Section V). Tables 6–8 summarize the most important findings concerning androgen action in male and female rats, respectively.
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B. Skeletal consequences of gonadectomy in rodents
1. Skeletal effects of gonadectomy in rats.
Surgical castration, as induced by ovx in the female rat and orch in the male rat, represents the most frequently used procedure to study skeletal sex steroid action. Chemical castration, as induced by GnRH agonists, has similar skeletal effects as surgical castration in female rats (37), but its impact on bone has not been studied in male rats. Both orch and ovx dramatically reduce serum levels of T and E2 in male and female rats. However, these procedures do not totally eliminate E2 production, because adrenal androgens can be transformed into estrogens after aromatization. In both genders, castration has considerable impact on cancellous and cortical bone compartments. However, whereas this response appears to be similar in cancellous bone, it is different in cortical bone.
Skeletal cancellous changes are characterized by an increase in cancellous bone turnover, resulting in bone loss in gonadectomized rats, irrespective of gender, age, or strain (203, 204, 205, 206, 207, 208, 209, 210, 211) (Table 6). Cancellous bone loss after gonadectomy can be detected not only by histomorphometry but also by peripheral quantitative computed tomography (pQCT) and microcomputed tomography. Biochemical markers of bone resorption (e.g., urinary deoxypyridinoline) and formation (e.g., serum osteocalcin) reflect the early increase of cancellous bone turnover after gonadectomy in both genders (40, 204, 209, 211, 212, 213, 214). These changes in the cancellous bone compartment are reminiscent of high-turnover osteoporosis after menopause (215) and explain why the ovx female and the orch male rat model have gained wide acceptance as animal models for osteoporosis (216). Also in line with observations in postmenopausal osteoporosis in humans, the number of osteoclasts is increased after ovx (39, 203, 207, 212, 217, 218) or orch (208, 210, 211, 219). Osteoblast number, surface, and mineral apposition rates are up-regulated in an attempt to fill the increased number of resorption cavities created by these osteoclasts (203, 208, 210, 211, 220). Although relative changes in histomorphometric and biochemical indices of bone resorption and formation indices cannot predict final outcomes on cancellous bone mineral content, orch-induced bone resorption tends to be increased more and longer than formation, even at the level of the individual remodeling unit (211, 220). Therefore, and despite increased bone formation at the tissue level, net cancellous bone loss occurs as a result of an imbalance with bone resorption exceeding bone formation in each bone mineral unit. The microanatomical mechanism responsible for this cancellous bone loss after orch in rats is a reduction in trabecular number (33, 211, 220) and thickness (211).
In contrast to the human skeleton, the rodent cortical skeleton has no Haversian canals, which explains why intracortical bone loss is not readily observed in ovx and orch rodents. Some studies have reported increased cortical porosity in older orch rats (205, 221), probably due to orch-induced medullary expansion through increased resorption at the endocortical site. In contrast to the changes in cancellous bone, the responses to castration at the periosteal site and growth plate are essentially sexually dimorphic, especially in young growing rats (Table 6). Periosteal and longitudinal bone formation appear to be increased in some (40, 57, 58, 222, 223, 224), but not all (203, 204, 207), studies on ovx and decreased in the orch model (207, 210, 213, 219, 224) (Fig. 4). The end result will be cortical bone expansion and increased bone length in the ovx female but decreased cortical bone volume and bone length in the orch male. This increase/decrease in cortical bone volume after gonadectomy (in female and male rats, respectively) is primarily due to a relative increase/decrease in periosteal bone formation and not (or to a lesser extent) to significant changes of the inner endocortical perimeter (225). The implication is that sex hormone deficiency may have a more severe impact on the integrity of the male skeleton compared with the female skeleton. Gender-related differences in the response of periosteal bone to gonadectomy also explain why methods that measure areal rather than volumetric bone density, such as DXA, tend to overestimate bone loss in the orch male while underestimating bone loss in the ovx female. Moreover, cortical bone loss in the orch rat model, in contrast to changes in cancellous bone, is due to failure to gain new bone and not to net bone loss. These gender-specific responses in cortical bone volume are particularly important during active growth but less significant in elderly, slow-growing rats (209, 220) (Table 6). In older rats, longitudinal bone growth does not decrease after orch (205, 206, 209).
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, castration induces postmenopausal-like bone loss in the cancellous bone compartment in both genders, irrespective of age or strain of the rat. The intracortical bone compartment is relatively resistant to surgical castration, whereas the responses at the periosteal site and the growth plate appear to be sexually dimorphic, especially in younger rats, with androgens stimulating and estrogens inhibiting periosteal bone expansion.
2. Skeletal effects of gonadectomy in mice.
Reduced cortical bone growth, as described in rats, has also been observed in male orch mice (70, 226, 227). Female mice, in contrast to rats, do not experience significant cortical bone expansion after ovx (228, 229).
In cancellous bone, both ovx (228, 229, 230, 231) and orch (66, 70, 188, 227, 231) induce bone loss. This cancellous bone loss is associated with elevated markers of bone turnover (70, 229), and ovx and orch mice are therefore increasingly being accepted as animal models for the study of steroid action in postmenopausal-like osteoporosis (139).
C. Skeletal effects of androgen replacement in rodents
1. Skeletal effects of androgen replacement in rats
a. Effects of aromatizable and nonaromatizable androgens.
The aromatizable androgen T is a very effective bone-sparing agent. T not only fully prevents cancellous bone loss in orch rats (208, 209, 221) but is also bone-sparing in ovx rats (232), even at subphysiological concentrations (233) (Fig. 5) and irrespective of age. In addition, T antagonizes periosteal expansion in ovx rats (234), but increases periosteal bone formation in orch rats (209, 221, 234) (Table 7). Weaker aromatizable androgens, such as DHEA (223, 235) and androstenedione (218, 236), are evidently bone-sparing in the ovx rat model but have not been studied in male orch rats (Table 7). In the ovx rat model, bone protection by these androgens is exerted through the AR and not the ERs, as illustrated by the fact that the effect is blunted by concomitant administration of AR antagonists but not by aromatase inhibitors or ER antagonists (218, 235, 236).
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). However, DHT appears to be less effective than T in the elderly orch rat model (208, 237), in particular on cortical bone (Fig. 5). In this model, higher doses of DHT than T are indeed needed to obtain at least some bone-protective action on cancellous bone, but this occurs at the expense of side effects like hypertrophy of the ventral prostate and seminal vesicles. Moreover, high-dose DHT fails to prevent orch-induced cortical thinning (237). In conclusion, in both ovx female and orch male rats, aromatizable and nonaromatizable androgens show bone-protective action, especially at cancellous bone sites. Nonaromatizable androgens seem to be less effective than aromatizable androgens. A possible explanation for the relative lack of efficacy of nonaromatizable androgens compared with aromatizable androgens may be the aromatization of the latter via estrogens and stimulation of the ERs.
b. Effects of estrogens.
The bone-sparing effects of aromatizable androgens may depend on activation of the AR, the ERs, or both. Estrogens (including phytoestrogens) have well-documented bone-sparing effects, not only in ovx rats (204, 232, 235, 239, 240, 241, 242) but also in orch rats (209, 213, 237) (Table 7).
Overall, in gonadectomized rat models, aromatizable and nonaromatizable androgens and estrogens (Table 7) appear to be bone-sparing in the cancellous bone compartment, irrespective of gender or age. Table 7 also indicates that sex steroid action on cortical bone is not only less well documented but also less consistent. Both androgens and low-dose estrogens tend to stimulate cortical bone, however only in male rodents.
2. Skeletal effects of sex steroids in mice.
T effectively prevents cancellous bone loss in orch mice (70, 188). DHT (96) and estrogens (66, 70, 96, 228, 231) also appear to be bone-sparing after castration, in both genders. Similar bone-sparing effects have been observed with phytoestrogens in ovx (243) and orch mice (244). So, in accordance with experiments in rats, androgens and estrogens both protect against cancellous bone loss in mice, irrespective of gender. Additionally, T and estrogens increase the cortical area in orch mice (226, 245).
D. Skeletal effects of selective manipulation of estrogen and androgen action in rodents
1. Effects of selective pharmacological modulation in rats.
Administration of AR antagonists, ER antagonists, SERMs, and aromatase inhibitors will selectively interfere with the AR, the ERs, and the aromatization of androgens into estrogens, respectively (Fig. 1).
Aromatase inhibitors impair both skeletal development (cortical expansion) and maintenance (integrity of the cancellous compartment) in male rats (47, 48) (Table 8). The bone-phenotypic changes induced by administration of an aromatase inhibitor are thus similar to those observed after orch, although bone turnover seems to be less elevated. Moreover, E2 prevents bone loss induced by an aromatase inhibitor in male rats, supporting the concept that pharmacological administration of estrogen is protective for male bone (49).
Administration of a selective ER antagonist, ICI 182,780, which induces cancellous bone loss in female intact and estrogen-repleted ovx rats (40, 57, 58), does not impair skeletal homeostasis in T-supplemented orch rats (59) or intact male rats (60) (Table 8). Yet, SERMs, some of which are being used in the treatment of postmenopausal osteoporosis, have been reported to restore ovx-induced bone loss in female rats (246, 247) (Table 7) as well as orch-induced (Table 7) and age-related bone loss (Table 8) in male rats (248, 249). Similarly, tibolone, a drug with mixed androgenic, estrogenic, and progestogenic properties, prevents bone loss in ovx rats (212) (Table 7). The bone-sparing effect of tibolone is reversed by concomitant administration of an antiestrogen but not by an antiprogestogen or antiandrogen, suggesting that bone protection by tibilone after ovx occurs through the ER pathway (232).
The skeletal effects of AR antagonists in rats are not consistent (Table 8). In older studies, AR antagonists like flutamide or cyproterone acetate were found to induce bone loss in rats, irrespective of gender; however, in these studies, bone mass was only assessed by bone calcium content or kinetics (36, 37). Several histomorphometric studies have reported significantly reduced bone formation or an increase in bone resorption after AR antagonist administration, but without concomitant bone loss (38, 39, 40). Confirmation of AR antagonist-induced osteopenia by methods such as histomorphometry is therefore required. Finally, finasteride, a type II 5-reductase inhibitor that blocks the conversion of T into DHT, does not interfere with skeletal homeostasis in male rats (33) (Table 8).
In conclusion, selective modulation of the AR and ER pathways generally supports the concept of a dual mode of androgen action (through both the AR and ER pathways), at least in cancellous bone, both in male and female rat models (Table 8). Effects of AR antagonists in rats, on the other hand, are less well-established.
2. Effects of selective pharmacological modulation in mice.
In intact male mice, the AR antagonist casodex does not affect bone density, whereas another AR antagonist, epitestosterone, which is also a 5-reductase inhibitor, decreases bone density (250). The AR antagonist cyproterone acetate prevents orch-induced bone loss, suggesting that this agent may act as an AR agonist in bone (251). SERMs also prevent orch-induced (227, 231) and ovx-induced bone loss (231, 252) in mice. Overall, these studies (although limited in number) provide further evidence for a similar action of sex steroids in cancellous bone. Along these lines, the activator of nongenomic estrogen-like signaling, estren, has a similar (or even greater) anabolic effect on (especially cortical) bone than DHT in male orch mice or than E2 in female ovx mice, despite a much lower affinity for the ER (96) (see also Section II). These effects of estren, although obtained with a much higher dose than with E2 and DHT, occur without stimulation of reproductive organs in either sex.
E. Skeletal effects of selective manipulation of androgen and estrogen action in transgenic mice
1. Description of transgenic animal strains.
The cyp19 aromatase gene has been inactivated [aromatase knockout (ArKO) mice] by two independent groups (253, 254), and a similar skeletal phenotype has been reported (54, 55, 56). ERß has been inactivated by three independent groups [ERß knockout (BERKO) mice] (79, 255, 256), and the skeletal phenotypes of all three mouse strains are rather similar (64, 65, 66, 67, 73, 75, 76, 77, 78, 79, 80, 81, 98). Two independent groups have inactivated ER [ER knockout (ERKO) mice] (256, 257). The first and most studied ERKO mouse strain was generated in the laboratories of Korach and Smithies (257). A recent study indicates that these mice are not completely ER-inactivated (258), as supported by the observation that they express one or two N-terminally modified ER transcripts associated with minor ER activity regarding uterine weight and endothelial nitric oxide production (258). The remaining ER activity is suggested to be mediated via a truncated ER with remaining AF-2 activity, whereas there is no AF-1 activity left (258). This truncated ER isoform has been detected in bone of these mice and has been shown to have effects on gene transcription in cultured human osteoblasts (259). These mice will be referred to as ERKOAF-1–/– because they do not have any remaining AF-1. The second ER-inactivated mouse model was developed in the laboratory of Chambon (256). There is no remaining ER activity in these mice, and both AF-1 and AF-2 activity are absent in these animals, which will be referred to as ERKOAF-1/AF-2–/–. The skeletal phenotype has been reported for both the ERKOAF-1–/– mice (62, 63, 64, 65, 66, 67, 69, 260, 261) and the ERKOAF-1/AF-2–/– mice (46, 71, 72, 73, 74, 98). Most skeletal phenotypes (including the male skeletal phenotype and the skeletal responses to estrogen treatment in female ovx mice) are identical for the ERKOAF-1–/– (66, 67, 70) and the ERKOAF-1/AF-2–/– mice (72, 73). However, a clear difference between the two ERKO models is seen in female gonadal intact mice (see Section IV.E., 2and 3). Possible factors that might explain the differences between the two ovarian intact female ERKO models include: 1) differences in genetic background; 2) differences in dietary/environmental estrogen; 3) differences in the feedback regulation of sex steroids between the ERKOAF-1–/– and ERKOAF-1/AF-2–/– mice, in which both E2 and T are dramatically increased but the magnitude of this disturbance might differ between the two ERKO models; 4) remaining AF-2 activity in ERKOAF-1–/– mice; and 5) other unknown reasons.
Mice with inactivated ER and ERß [double ER knockout (DERKO)], developed in two independent laboratories, have been reported to have rather similar skeletal phenotypes, both in intact mice and in estrogen-treated gonadectomized mice (64, 65, 66, 67, 72, 73, 98). AR knockout mice (ANDRKO mice) have recently been developed by two independent groups using the cre/loxP system (42, 43, 44), and their skeletal phenotypes have recently been described (42, 43, 44, 262, 263).
It is of importance to note that the sex steroid levels (both E2 and T) are increased in ERKO and DERKO, due to disturbed feedback regulation (65, 73, 264). In contrast, the skeletal phenotype of single ERß inactivation is not influenced by altered sex steroids levels. By comparing the skeletal phenotype of wild-type, ArKO, BERKO, ERKO, DERKO, and ANDRKO mouse strains, it may be possible to gain new insight in the relative importance of the AR, ER, and ERß in mediating the skeletal effects of androgens. The different transgenic mouse strains described in this section are inactivated from birth, which results in disturbed skeletal growth and maturation. Thus, the adult skeletal phenotype in these mice is a combination of effects on growth/maturation and adult bone remodeling. Future transgenic mouse models with inducible gene inactivation will be useful in separating the effects on growth/maturation from the effects on adult bone remodeling and will therefore be more relevant to further explore the protective effect of sex steroids on the adult skeleton. Nevertheless, the currently available transgenic mouse models have already been very informative. In the following section, the effects on longitudinal appendicular skeletal growth as well as the effects on cortical and cancellous bone in these mice will be discussed.
2. Longitudinal appendicular skeletal growth
a. Females.
ERß-inactivated female mice develop increased femoral length after sexual maturation (65, 73, 75, 78). Actually, the normal length difference between males and females is not observed in young adult female BERKO mice (65, 75, 76, 78) (Fig. 6A). An altered length of femur is often associated with a disturbed GH-IGF-axis (265, 266, 267, 268). Interestingly, serum levels of IGF-I are increased in young adult female B
) and ovx (•) rats are shown as a function of time after ovx. P < 0.01 for all ovx time points compared with intact controls. B, The effect of orch on periosteal bone formation rate. The mean ± SE and tetracycline labeling period for intact controls (
) and orch (
) are shown as a function of time after orch. P < 0.01 for all orch time points compared with the same labeling period in intact controls. [Reproduced from R. T. Turner et al.: J Orthop Res 8:612–617, 1990 (