Minireview: Osteoprotective Action of Estrogens Is Mediated by Osteoclastic Estrogen Receptor-α

Abstract

The osteoprotective action of estrogen in women has drawn considerable attention because estrogen deficiency-induced osteoporosis became one of the most widely spread diseases in developed countries. In men, the significance of estrogen action for bone health maintenance is also apparent from the osteoporotic phenotype seen in male patients with genetically impaired estrogen signaling. Severe bone loss and high bone turnover, including typical osteofeatures seen in postmenopausal women, can also be recapitulated in rodents after ovariectomy. However, the expected osteoporotic phenotype is not observed in female mice deficient in estrogen receptor (ER)-α or -β or both, even though the degenerative defects are clearly seen in other estrogen target tissues together with up-regulated levels of circulating testosterone. It has also been reported that estrogens may attenuate bone remodeling by cell autonomous suppressive effects on osteoblastogenesis and osteoclastogenesis. Hence, the effects of estrogens in bone appear to be complex, and the molecular role of bone estrogen receptors in osteoprotective estrogen action remains unclear. Instead, it has been proposed that estrogens indirectly control bone remodeling. For example, the enhanced production of cytokines under estrogen deficiency induces bone resorption through stimulation of osteoclastogenesis. However, the osteoporotic phenotype without systemic defects has been recapitulated in female (but not in male) mice by osteoclast-specific ablation of the ERα, proving that bone cells represent direct targets for estrogen action. An aberrant accumulation of mature osteoclasts in these female mutants indicates that in females, the inhibitory action of estrogens on bone resorption is mediated by the osteoclastic ERα through the shortened lifespan of osteoclasts.

Estrogen is osteoprotective by attenuating bone resorption in females and males due to hormonally induced death of mature osteoclasts.

Estrogen is a prime female steroid hormone as well as a pivotal regulator in many biological processes beyond development and maintenance of female reproductive organs. Among the estrogen target organs, bone has recently drawn increasing attention because postmenopausal osteoporosis induced by estrogen deficiency has emerged as the most widely spread bone/joint disease in developed countries. Osteoporosis in women and men is currently considered a serious disorder of middle-aged and elderly people because of increased risk of bone fracture, often leading to long-term incapacitation and high mortality (1, 2).

Pronounced bone mass decrease due to enhanced or imbalanced bone resorption vs. bone formation (high bone turnover) is a typical osteoporotic feature in women with estrogen deficiency or impaired estrogen signaling (Fig. 1). The osteoporotic bone phenotype can be experimentally recapitulated in female rodents by ovariectomy (OVX) and consequent estrogen depletion (3, 4). Bioavailable estrogens and selective estrogen response modulators are shown to be effective at attenuating high bone turnover and prevent bone loss in both osteoporotic patients and OVX rodents (3, 4, 5, 6). Accumulating clinical observations and genetic studies show that male patients defective in either estrogen biosynthesis or function of estrogen receptor α (ERα) display typical pathological conditions of osteoporosis (7, 8). Thus, it is evident that estrogens exert osteoprotective actions and play a significant role in skeletal maintenance in both sexes.

Fig. 1.

The role of bone cells in bone remodeling. Bone tissue is continuously remodeled by osteoblastic bone formation and osteoclastic bone resorption. The balance between bone formation and resorption is maintained under normal physiological conditions (upper panel). However, when this balance is altered, pathological conditions such as osteoporosis may develop in which increased osteoclastic bone resorption exceeds osteoblastic bone formation with a resultant decrease in bone mass (lower panel).

The first conventional gene disruption of the mouse ERα locus was achieved in the early 1990s (9). Paradoxically, however, neither males nor female ERα-deficient mice exhibited typical osteoporotic bone phenotypes (10, 11). Thereafter, the role of ERs in bone health remained obscure. Instead, indirect mechanisms via extraskeletal tissues have been postulated to account for the osteoprotective actions of estrogen (12, 13). In this review, we will describe the skeletal and extraskeletal activities of ERs in mediating osteoprotective estrogen actions.

Nuclear ERs

Both subtypes of nuclear ERs, α and β, are members of the nuclear steroid hormone receptor gene superfamily and mediate most biological effects of estrogens (9, 14, 15). Nuclear estrogen-bound ERs are responsible for the genomic actions of estrogen through estrogen response element-dependent transcriptional control of target genes (16, 17, 18, 19) (Fig. 2). Rapid estrogen responses, so-called nongenomic actions, likely require cytoplasmic ERs and/or uncharacterized atypical ERs on the cell membrane (20).

Fig. 2.

The molecular mechanism of gene regulation by sex steroid hormones. Sex steroid hormones bind to their cognate nuclear receptors, and the ligand-bound receptors act as transcription factors regulating expression of specific target genes. Testosterone (weak androgen) can be converted by 5α-reductase into an active form of androgen, dihydrotestosterone (DHT). At the same time, testosterone serves as a precursor for an active estrogen, 17β-estradiol, through conversion by aromatase.

Both ERα and ERβ recognize and specifically bind to estrogen response elements in the target gene promoters as homodimers (α-α or β-β) and/or heterodimers (α-β) (9, 14, 15, 21). No clear difference in the binding of endogenous estrogens has been observed between ERα and ERβ; however, the ERs appear to exhibit different affinities for selective estrogen response modulators (22). In comparison with ERα, ERβ apparently has a lower capacity for hormone-induced transcriptional activation (23). Therefore, ERβ can be thought of as a dominant-negative counterpart of ERα that moderates the induction of endogenous estrogen target genes as well as transcriptional responses to estrogens. The molar, or quantitative, ratio of ERβ to ERα in a given cell is thus considered to define the cell’s sensitivity to estrogens and the extent of its biological responses to the hormone (24). Expression patterns of ERα and ERβ overlap in many organs and tissues, whereas in some types of cells, only one ER subtype is detectable.

ER-Deficient Female Mice Display Expected Abnormalities in the Estrogen Target Tissues But Not in Bones

The first generated ERα knockout (ERαKO) mice exhibited a wide spectrum of phenotypic abnormalities. Consistent with previously accumulated in vitro and in vivo findings, female reproductive organs in these ERαKO mice were poorly developed, and their phenotypes closely resembled features of OVX mice (9). However, the first ERαKO reportedly expressed shortened ER transcripts, and a residual ERα activity was suspected to mediate the endothelial effects of estrogens (25). Later, complete ERαKO mice (ERα^−/−) expressing no detectable ERα transcripts were generated. Phenotypes of the ERα^−/− mice verified and confirmed the ERα impact on female reproductive organs reported earlier in the ERαKO mice (26). Mice with a disrupted ERβ gene have been obtained by several groups; however, the described phenotypes of independently generated ERβ-deficient mice have not been consistent in the resultant abnormalities of estrogen target tissues (11, 24). Inconsistency between these initial studies was apparently based on analyses of mice with incomplete knockout of the ERβ (ERβKO) that could express abnormal transcripts. The phenotypic difference could be also due to residual ERβ activity, but the molecular basis of the varied observations still remains unclear at this stage. Interestingly, fewer abnormalities have been observed in recently generated complete ERβKO (ERβ^−/−) mice (26, 27). Because ERα^−/− mice exhibit far more severe abnormalities in estrogen target tissues than ERβ^−/− mice (9, 10, 11, 27, 28), it appears that ERα is the prime receptor mediating major physiological actions of estrogens (Table 1).

Table 1.

Phenotypes of ERKOs in reproductive organs

	ERαKO	ERβKO	ERαβKO
Pituitary gland	High LH	Normal	High LH
				Hemorrhagic cystic
Ovary	High estrogen	Reduced ovulation	Lack of ovulation
	High testosterone		Sex-reversed follicles
	Anovulatory
Uterus	Estrogen in sensitive	Normal	Estrogen insensitive
Mammary gland	No pubertal development	Normal	No pubertal development

Unlike other estrogen target tissues, female bones were not significantly affected by depletion of either ERα (ERα^−/−) or ERβ (ERβ^−/−) or both (ERαβ^−/−), and the bone phenotypes were quite mild (29, 30, 31, 32). It was unexpected, because OVX in the same mouse strains led to a decrease in bone mass with high bone turnover that resembled the bone phenotype in women with naturally occurring estrogen deficiency in a postmenopausal state (1, 3, 13, 33).

The Osteoporotic Phenotype Is Absent in Female Mice Deficient of ERs

Lower bone turnover and increased bone mass were seen in female ERα^−/− mice, whereas OVX in the same mouse strain induced decreased bone mass due to increased bone resorption (30, 32). The lack of negative feedback regulation due to the absence of ERα in the pituitary in ERα^−/− mice results in aberrantly high serum levels of testosterone, a major estrogen precursor. The absence of osteoporotic phenotypes in ER-deficient mice can be explained by the osteoprotective effects of enhanced androgen signaling by testosterone excess in the bone (33). Supporting this idea, OVX induces bone loss in the ERα^−/− mice, whereas androgen administration has osteoprotective effects in OVX mutant females (34, 35).

ERβ^−/− female mice also exhibit reduced bone resorption and increased trabecular bone volume without, however, alteration of circulating sex hormone levels (32). Because estradiol can still be effective at partially reversing bone loss in OVX ERα^−/− females (35), it appears that ERβ also mediates the osteoprotective action of estrogen, but to a much lesser extent than ERα (11, 35).

In the double-knockout (ERαβ^−/−) mice, levels of circulating sex steroid hormones were also elevated, similar to those in ERα^−/−. However, unlike the ERα^−/− or ERβ^−/− mice, a marked bone mass decrease was observed in the ERαβ^−/− mice (Table 2), but it was less pronounced than that observed in the OVX wild-type females (30, 32, 34). Although these findings suggest cooperation between ERα and ERβ in the osteoprotective action of estrogens, female mice deficient in both ERα and ERβ fail to recapitulate the osteoporotic bone phenotype of estrogen-deficient women. It has been proposed that at high levels of circulating sex steroids, androgen receptor (AR) may partially compensate for ER deficiency in these mice.

Table 2.

Summary of ERKO bone phenotypes and levels of sex steroid hormones

	ERαKO			ERβKO			ERαβKO			OcERαKO
		Male	Female	Male	Female	Male	Female	Male	Female
Trabecular BMD in young	↓	→	→	→	↓	↓	→	↓
Trabecular BMD in old	↑	↑	→	↑	↑	→	→	↓
Cortical BMD	↓	↓	→	→	↓	↓	→	→
Trabecular BV/TV	↑	↑	→	↑	↑	↓	→	↓
Bone formation	↓	↓	→	→	↓	↓	→	↑
Bone resorption	↓	↓	→	↓	↓	→	→	↑
17β-Estradiol	→ or ↑	⇈	→	→	→	→ or ↑	→	→
Testosterone	⇈	↑	→	→	⇈	→ or ↑	→	→

BMD, Bone mineral density; BV, bone volume; TV, trabecular volume.

Can Local Aromatase-Produced Estrogens Support Normal Bone Remodeling in Males?

Male mice deficient of one (ERα^−/− or ERβ^−/−) or both (ERαβ^−/−) of the ER subtypes exhibited neither clear bone mass decrease nor impaired bone development. Moreover, ERα^−/− males displayed an increase in trabecular bone and decrease in bone resorption with elevated levels of testosterone (30, 32, 34) (Table 3). It has been suggested that the higher level of circulating testosterone in ERα^−/− males compensates for impaired estrogen action by enhancing AR function in the bone (35, 36). These observations are, however, inconsistent with severe osteoporosis, and unfused epiphyses seen in men with nonfunctional ERα owing to hereditary mutations and despite high (albeit normal for men) levels of circulating testosterone (7, 9, 10, 37) (Table 3). Consistently, these male patients have failed to respond to estrogen treatment. The idea that ERα-mediated estrogen signaling is involved in the maintenance of the male skeleton is further supported by clinical observations that bone mass is low in aromatase-deficient male patients (8, 38, 39) (Table 3). Aromatase (CYP19) is the enzyme catalyzing conversion of androgens into estrogens (Fig. 2). Because circulating estrogen levels are low in males, the local conversion of testosterone into estrogen by aromatase appears to be physiologically significant for osteoprotective estrogen action in male bones. Reduced bone mass, high bone turnover, and unfused epiphyses, similar to those in ERα-deficient men (7), have also been observed in aromatase-deficient male patients (8, 38, 39). Significantly, such osteoporotic bone defects could be rescued by estrogen treatment only in aromatase-deficient, but not ERα-deficient, patients (40). Bone mass decrease was experimentally recapitulated in male mice deficient of aromatase (41, 42), whereas femur growth acceleration due to unclosed epiphyses in human male patients was not detected in male aromatase-deficient mice, presumably owing to innate absence of epiphyseal plate closure in mice of both sexes (8, 37, 42). Thus, the bone defects seen in naturally occurring aromatase-deficient men as well as experimentally generated aromatase-deficient male mice suggest that estrogens locally produced by aromatase are beneficial for bone health in males.

Table 3.

Serum hormone levels and bone phenotypes in male ERα- and aromatase-deficient patients

	ERα deficiency	Aromatase deficiency
Estradiol	High	Low
Testosterone	Normal	Normal
FSH	High	High
LH	High	Normal
Bone mineral density	Low	Low
Bone formation marker	High	Normal
Bone resorption marker	High	Normal
Estrogen sensitivity	No	Yes
Others	Tall stature, unfused epiphysis	Tall stature, unfused epiphysis

Indirect Osteoprotective Action of Estrogens in Males and Females

Absence of the expected osteoporotic defects in male and female mice lacking ERs suggests an indirect mode of the osteoprotective action of estrogens. Namely, bone marrow and some extraskeletal estrogen target cells and tissues secrete endocrine or paracrine factors known to support bone development and remodeling (Fig. 3). It has been proposed that these estrogen-induced antiresorptive factors may mediate the osteoprotective effects of estrogens. IGFs, known to stimulate osteoblastogenesis, are secreted by the liver. They modulate GH action in bone development, particularly during growth stages, and in cultured bone cell systems (37, 43, 44). Thus, hepatic IGF production stimulated by estrogens has been assumed to account for the osteoprotective effects of estrogen. However, the molecular basis of the estrogen-induced IGF regulation remains to be determined.

Fig. 3.

Indirect actions of estrogen control bone remodeling. Estrogen production by the ovaries is stimulated by FSH secreted by the pituitary gland. Estrogens inhibit FSH secretion through negative feedback regulation mediated by the pituitary ER. Estrogen deficiency increases FSH secretion and stimulates production by bone marrow and immune cells of cytokines, such as TNFα and several ILs. These cytokines facilitate osteoclast differentiation from hematopoietic stem cells. As an opposing action, ovarian estrogens and GH from the pituitary gland induce hepatic production of IGF-I that stimulates osteoblast differentiation.

FSH reportedly mediates indirect estrogen action in bone mass maintenance as a negative bone remodeler (13, 45). FSH is an upstream pituitary hormone that stimulates estrogen production by the ovary. FSH secretion is under negative feedback control of circulating estrogens that activate ERs in the pituitary to suppress FSH production. Estrogen deficiency in postmenopausal women is often associated with increased levels of circulating FSH. Zaidi et al. (45) applied this epidemiological observation to a mouse genetic approach using mice deficient in either FSH or FSH receptor. Female mice in which FSH signaling was defective displayed increased bone mass and low bone turnover without significant alteration of serum estrogen levels. Because this group also showed FSH’s ability to stimulate osteoclastogenesis, they inferred that increased FSH levels stemming from estrogen deficiency might cause a decrease in bone mass through enhancement of bone resorption by increased osteoclastogenesis (45). However, it is problematic to experimentally assess the significance of FSH’s action in the overall osteoprotective action of estrogens under normal physiological levels of the sex steroid. Moreover, it has been reported that GnRH agonist-induced estrogen deficiency causes dramatic bone loss despite significant suppression of FSH production (46). Therefore, it appears unlikely that FSH exerts beneficial effects on the bone at optimal levels of estrogens, or in an estrogen-sufficient state.

Estrogen deficiency-induced osteoporosis associates with persistent imbalance between bone resorption and bone formation activities that leads to progressive bone mass reduction. This suggests that osteoclastogenesis and/or osteoclastic function are augmented under suboptimal estrogen concentrations. Because inflammatory cytokines are potent inducers of osteoclastogenesis in vitro, a number of cytokines expressed by nonbone cells have been tested for possible contributions to estrogen deficiency-induced osteoclastogenesis (12, 47, 48) (Fig. 3). In this respect, hematopoietic cells in the bone marrow have emerged as modulators of bone remodeling through secretion of various pro- and antiresorptive cytokines (12). Among circulating blood cells, T cells are drawing much interest as potential regulators of bone resorption. Estrogen deficiency activates adaptive immune responses leading to stimulation of IL-7 and IGF-I production by activated T cells residing in bone that further induces secretion of interferon-γ (49, 50). Interferon-γ acts as an osteoclastogenic factor in concert with locally produced receptor activator of nuclear factor-κB ligand and tumor necrosis factor (51, 52). In cell culture systems, these cytokines activate transcriptional factors activation protein-1 and nuclear factor-κB that are known to promote osteoclastogenesis. Thus, loss of bone mass during estrogen deficiency can be attributed, at least in part, to a consequent increase of proresorptive cytokine production (12). However, these cytokine effects only reflect enhancement of osteoclast activity subsequent to estrogen deficiency and do not provide insight into the molecular basis of the beneficial action of estrogens on bone remodeling.

Osteoclastic ERα Mediates the Osteoprotective Action of Estrogens in Females

As outlined above, studies of systemic estrogen deficiency and impaired estrogen signaling in experimental animal models failed to indentify the mechanisms of osteoprotective actions of estrogens. Indeed, complete elimination of estrogen signaling in the whole organism (e.g., by conventional knockout of the ER genes) causes systemic imbalance in the endocrine system as well as possible nonphysiological events like aberrant cytokine production (12, 13). Thus, it became apparent that a bone cell-specific disruption of the ER genes is required to directly assess the role of estrogen signaling in the bone without interference from its systemic action or potential secondary defects.

The osteoprotective action of estrogens is associated with attenuation of bone resorption and restoration of normal bone remodeling. We therefore selectively disrupted the ERα gene in mature osteoclasts (53). Because cathepsin K is expressed only in the developed osteoclasts (54), the Cre gene was knocked into the cathepsin K gene locus, and the resulting Cre knock-in transgenic line (CatK-Cre) was shown to express Cre at detectable levels only in mature osteoclasts. Osteoclast-specific ERαKO mice (ERα^ΔOC/ΔOC) were generated by crossing mice from the CatK-Cre line with mice from the floxed ERα gene line that was previously used to obtain the complete ERαKO mice (ERα^−/−) (26). As expected, neither clear alterations of circulating sex steroids nor FSH nor phenotypic abnormalities in growth and reproduction were detectable in male or female ERα^ΔOC/ΔOC mice. At 8 wk of age, significant bone loss in the trabecular bone area with high bone turnover was observed in the ERα^ΔOC/ΔOC females, but not males (Table 2). OVX caused only a negligible bone loss in the ERα^ΔOC/ΔOC mutants when compared with the bone loss in OVX wild-type females. In ERα^ΔOC/ΔOC females, 17β-estradiol effectively restored bone mass in the cortical area but not in the trabecular area. Considering that bone loss becomes evident first in the trabecular area in estrogen-deficient female rodents and in women, the osteoporotic features observed in the ERα^ΔOC/ΔOC female appear to support the idea that the osteoclastic ERα mediates, at least in part, the osteoprotective action of estrogens in females (53). Because estrogens can trigger apoptosis in osteoclasts through induction of the Fas ligand (FasL) gene, we suggest that the proapoptotic actions of estrogens in mature osteoclasts underlie the antiresorptive effects of estrogens in the bone (53) (Fig. 4). These findings are consistent with earlier observations that OVX-induced estrogen deficiency in mice resulted in decreased apoptosis and extended lifespan of mature osteoclasts (55).

Fig. 4.

Proposed model of the osteoclastic ERα-mediated osteoprotective action of estrogens through attenuation of bone resorption by inducing osteoclastic cell death. Estrogens (17β-estradiol) activate ERs in functionally opposing types of bone cells: osteoblasts, which derive from mesenchymal stem cells, and osteoclasts, multinucleated giant cells derived from hematopoietic stem cells. The estrogen-dependent induction of FasL gene expression in these bone cells triggers apoptosis in mature osteoclasts.

Male ERα^ΔOC/ΔOC Mice Display No Apparent Bone Defects

Contradictory to expectations raised by previous studies in rodents and male patients (8, 29, 41, 42), no abnormality has been observed in either cortical or trabecular bone areas in ERα^ΔOC/ΔOC males (53). It is difficult to ascertain the reason why ERα^ΔOC/ΔOC males do not exhibit bone loss, and here we can only speculate about a possible difference in mechanisms of osteoprotective estrogen actions in males. First, high levels of androgens, and consequently, activated AR may critically impact bone formation and resorption even in the absence of ERα. Second, the CatK-Cre transgenic line is limited to disrupt a given gene only at late stages of osteoclastic life, whereas the activated ERα may play a critical role at earlier stages. There may be stage-specific differences between males and females in the ERα action during osteoclast differentiation and maturation. This idea is supported by previous reports that estrogens may be inhibitory for osteoclastogenesis and osteoclastic function in vitro (56, 57). Generation of differentiation stage-specific osteoclastic ERα knockout mice may be able to clarify this hypothesis. Thirdly, osteoblastic ERα may also mediate the osteoprotective effects of estrogens. Earlier, estrogens were shown to stimulate osteoblastogenesis in vitro (58). More recently, Brown’s group provided evidence that the FasL gene is a direct ER target in cultured osteoblasts (59). It is thus conceivable that estrogens support apoptosis of mature osteoclasts via stimulation of FasL expression in osteoclasts and osteoblasts (60). Reported earlier antiapoptotic effects of estrogens on osteoblasts (61, 62) suggest the existence of differential mechanisms or even pathways of estrogen action in these two cell types. Thus, it is conceivable that, in contrast to proapoptotic effects of estrogens on osteoclasts, their antiapoptotic effects on osteoblasts are similar in males and females. Obviously, osteoblast-specific ablation of ERs in male mice is required to challenge or prove this idea. Likewise, there are several cell types, including osteocytes and bone marrow cells, which remain to be tested for the impact of their ERs on the osteoprotective estrogen actions in males.

Conclusion

Estrogens exert osteoprotective actions in females and males. Key features of the estrogen deficiency-induced osteoporosis observed in postmenopausal women, such as bone loss and high-turnover bone metabolism, has been recapitulated in osteoclast-specific ERα knockout female mice (53). This is in striking contrast to the phenotype of mice with conventional or general ER knockout that exhibited increased bone mass. It appears that in females, osteoclastic ERα mediates estrogen-dependent attenuation of bone resorption through stimulation of apoptosis in osteoclasts. Although primary cultured bone cells from males and females equally respond to estrogen, the osteoclast-selective ablation of ERα in male mice caused neither bone defects nor unclosed epiphyses (53) that had been consistently observed in male patients with impaired estrogen signaling (7, 8). This discrepancy suggests sex specificity in mechanisms of osteoprotective action of estrogens in vivo and raises a hypothesis that in male bones, beneficial effects of estrogens are predominantly mediated by the osteoblastic ERα, rather than through the antiresorptive action of osteoclastic ERα, which is more critical in females. This idea can be tested by cell type-specific ablation of ERα in males as well as in females to decipher molecular and cellular mechanisms of the anabolic action of estrogens in the skeleton. At the same time, a compensatory action of AR at high concentrations of circulating testosterone may account for different physiological consequences of osteoclast-targeted ERα ablation in male mice. A double osteoclast-specific AR and ERα knockout may clarify this possibility.

NURSA Molecule Pages:

• Ligands: 17β-estradiol;

• Nuclear Receptors: ER-α | ER-β.