Regulation of Bone Metabolism by Sex Steroidsperspectivesinmedicine.cshlp.org/content/8/1/a031211... · 2017-12-20 · Regulation of Bone Metabolism by Sex Steroids Sundeep Khosla

Regulation of Bone Metabolism by Sex Steroids

Sundeep Khosla and David G. Monroe

Robert and Arlene Kogod Center on Aging and Endocrine Research Unit, Mayo Clinic College of Medicine,Rochester, Minnesota 55905

Correspondence: [email protected]

Osteoporosis is a significant public health problem, and a major cause of the disease isestrogen deficiency following menopause in women. In addition, considerable evidencenow shows that estrogen is also a major regulator of bone metabolism in men. Since theoriginal description of the effects of estrogen deficiency on bone by Fuller Albright morethan 70 years ago, there has been enormous progress in understanding the mechanisms ofestrogen and testosterone action on bone using human and mouse models. Although weunderstand more about the effects of estrogen on bone as compared with testosterone, bothsex steroids do play important roles, perhaps in a somewhat compartment-specific (i.e.,cancellous vs. cortical bone) manner. This review summarizes our current knowledge ofsex steroid action on bone based on human and mouse studies, identifies both agreementsand potential discrepancies between these studies, and suggests directions for future researchin this important area.

Osteoporosis is a significant and growingpublic health problem; in fact, the number

of women who will experience a fracture in oneyear exceeds the combined number of womenwho will experience incident breast cancer,myocardial infarction, or stroke (Cauley et al.2008). The major trigger for the developmentof osteoporosis in women is the onset ofestrogen deficiency following menopause(Riggs et al. 2002). Although men also developosteoporosis with aging, they lack the abruptcessation of gonadal function present inwomen. Nonetheless, considerable evidence in-dicates that even in men, declining sex steroidlevels with aging, particularly declining bio-available estrogen levels, contribute to age-relat-ed bone loss (Khosla et al. 2008). Thus, a betterunderstanding of how sex steroids regulate bone

metabolism is critical not only from a biologicalbut also a clinical perspective.

Seminal studies by Fuller Albright morethan 70 years ago showed that loss of estrogenfollowing oophorectomy or menopause led torapid bone loss and that calcium balance couldbe improved in postmenopausal women withestrogen treatment (Albright 1940). Decadeslater, Stepan and colleagues (1989) showedthat, following castration, men also experiencedsimilar rapid bone loss; because testosterone isthe major sex steroid in men, this led to thelongstanding dogma that estrogen was themajor regulator of bone metabolism in women,with testosterone playing the analogous role inmen. Subsequent studies established that com-plete sex steroid deficiency in either sex wasassociated with an activation of bone remodel-

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ing (bone resorption and bone formation), butwith a net deficit in bone formation relative tobone resorption and subsequent bone loss(Riggs et al. 2002). This review will delineatethe evolution of our understanding of sexsteroid regulation of bone metabolism, startingwith human clinical–investigative and observa-tional studies and then moving into data frommouse models. Although the human studieshave provided important insights into physiol-ogy and the pathogenesis of osteoporosis, themouse models have been instrumental in pro-viding key mechanistic insights. Nonetheless,there are discrepancies between the humanand mouse studies, and these will be highlightedwhere appropriate, with suggestions for poten-tial resolutions to disparate findings. Althoughthere is correlative evidence for relationshipsbetween weak adrenal androgens (Khosla et al.1998) or progesterone (Prior 1990) and bonemineral density (BMD), direct evidence frominterventional studies in humans is inconsistentfor these steroids, so the focus will be on estro-gen and testosterone effects on bone.

HUMAN STUDIES

Evidence from Estrogen Receptor– orAromatase-Deficient Males

The traditional notion that estrogen was thedominant sex steroid regulating bone metabo-lism in women, whereas testosterone played theanalogous role in men was challenged by thedescription in 1994 of the skeletal phenotypeof a 28-year-old male with homozygous muta-tions in the estrogen receptor (ER)a gene, re-sulting in a nonfunctional ER (Smith et al.1994). Despite normal testosterone and elevat-ed estrogen levels, this individual had unfusedepiphyses and osteopenia, with a spine BMDthat was more than two standard deviations(SDs) below the mean for 15-year-old boys(the patient’s bone age). Subsequent to this re-port, two males with complete deficiency of theenzyme responsible for the final step in the syn-thesis of estrogens, aromatase, were described(Carani et al. 1997; Bilezikian et al. 1998) witha virtually identical skeletal phenotype to the

ERa-deficient male. These “experiments of na-ture” clearly showed that, even in boys, estrogenwas essential for epiphyseal closure and for theacquisition of bone mass during puberty inmales. A subsequent description of a transiliaccrest bone biopsy of the ERa-deficient maleshowed reduced cancellous bone volume andcortical width (Smith et al. 2008). Interestingly,in contrast to estrogen-deficient women or hy-pogonadal men who typically have increasedbone remodeling (Riggs et al. 2002), the ERa-deficient male had markedly reduced indices ofbone resorption and formation. This suggeststhe possibility that loss of the ligand (estrogen)may lead to reduced bone mass through adifferent mechanism than loss of the receptor.Indeed, this possibility is supported by numer-ous ligand-independent effects of ERa in boneand other tissues (Ciana et al. 2003). However,cultured bone marrow stromal cells from theERa-deficient male also showed increasedERb levels by Western analysis (Smith et al.2008). The role of ERb in bone metabolism isdiscussed later in greater detail in the mousemodels, but at least in this human case reportthe high circulating estrogen and increasedbone ERb levels could not compensate for lossof ERa to preserve bone mass. Whether the lowbone turnover with loss of ERa was because ofthe absence of ligand-independent effects ofERa or some degree of compensatory suppres-sion of bone turnover because of the activationof ERb by the elevated circulating estrogenlevels remains unclear and warrants furthermechanistic studies using mouse models.

Observational Studies

Extensive studies in women through themenopausal transition have confirmed FullerAlbright’s original observations regarding thekey role of estrogen in regulating bone meta-bolism in women. Specifically, as shown inFigure 1, the decline in serum estradiol levels(and accompanying increase in serum folliclestimulating hormone [FSH] levels) throughmenopause is closely associated with an increasein osteoclastic bone resorption (urine N-telo-peptide of type I collagen [NTx]) (Sowers et

S. Khosla and D.G. Monroe

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al. 2013). Conversely, because the postmeno-pausal ovary continues to produce androgens,serum testosterone or other androgen levelsdo not change significantly across menopause(Handelsman et al. 2016), arguing against a rolefor androgens in contributing to postmeno-pausal bone loss. Moreover, the specific role, ifany, of the relatively low circulating androgenlevels in women in regulating bone metabolismhas been difficult to identify in human studiesand will be considered further in the mousemodels discussed later.

Numerous observational studies in menhave documented that, consistent with the find-ings from the ERa- and aromatase-deficientmales, serum estrogen levels were more closelycorrelated with BMD and bone turnover mark-ers than were serum testosterone levels. Using apopulation-based age-stratified sample of 346men aged 23–90 years (Khosla et al. 1998), wefound that serum total testosterone and estro-gen (estradiol þ estrone) levels decreased overthe life span by 30% and 12%, respectively, butthe bioavailable (or non-sex-hormone-bindingglobulin-bound) testosterone and estrogen lev-els decreased by 64% and 47%, respectively. Us-ing multivariate analyses, serum bioavailableestrogen level was the consistent independent

predictor of BMD in men. Thus, serum bio-available estrogen levels not only declined sig-nificantly with age in men, but also correlatedwith declining BMD levels, suggesting that es-trogen deficiency with aging may contribute tobone loss not only in women, but also in men.Since these original findings, numerous subse-quent analyses have confirmed the closer asso-ciation of BMD and rates of bone loss withserum bioavailable estrogen as compared withtestosterone levels in men (for review, seeKhosla et al. 2008).

Interventional Studies

The evidence from interventional studies isoverwhelming that estrogen is the dominantsex steroid regulating bone metabolism inwomen. The most rigorous demonstration ofthis comes from the results of the Women’sHealth Initiative, which randomized 16,608postmenopausal women aged 50 to 79 years toreceive conjugated equine estrogen (0.625 mg/d)and medroxyprogestorone acetate (to preventendometrial hyperplasia, 2.5 mg/d) or placebofor an average of 5.6 years (Cauley et al. 2003).Estrogen treatment increased BMD at multipleskeletal sites as compared with placebo andalso reduced hip fracture risk by 33%. In con-trast to estrogen, evidence that the low circulat-ing testosterone levels present in women have asignificant impact on bone metabolism is weakor inconsistent (Raisz et al. 1996).

To directly compare estrogen versus testos-terone effects on bone metabolism in men, weperformed an interventional study in whichendogenous estrogen and testosterone produc-tion were pharmacologically suppressed andphysiological levels of each were maintainedby exogenous replacement using estrogen andtestosterone patches (Falahati-Nini et al. 2000).Following baseline measurements of boneresorption (urinary deoxypyridinoline [Dpd]and NTx) and bone formation (serum osteocal-cin and amino-terminal propeptide of type Icollagen [PINP]) markers, the men were ran-domized into four groups: group A (2T, 2E)discontinued both patches; group B (2T, þE)discontinued the testosterone but remained on

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Figure 1. Changes in estradiol, follicle-stimulatinghormone (FSH), and bone resorption through themenopausal transition. Population mean bone re-sorption marker (urine N-telopeptide of type I col-lagen [NTx]), serum estradiol, and serum FSH levelsin relation to years from final menstrual period.Dashed lines denote the 95% confidence intervals.(From Sowers et al. 2013; reproduced, with permis-sion, from Oxford University Press # 2013.)

Sex Steroids and Bone

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the estrogen patch; group C (þT, 2E) discon-tinued the estrogen but remained on the testos-terone patch; and group D (þT, þE) continuedboth patches. As shown in Figure 2A, significantincreases in the bone resorption markers (Dpdand NTx) in the group with complete sexsteroid withdrawal (group A) were entirelyprevented by replacement of both sex steroids(group D) and almost completely prevented byestrogen replacement alone (group B). In con-trast, testosterone replacement in the absence ofconversion to estrogen (because all men werealso maintained on an aromatase inhibitor)was largely ineffective. A formal statisticalanalysis using a two-factor analysis of variance(ANOVA) model established a highly significanteffect of estrogen, but not testosterone, onbone resorption, and we estimated based onthese data that, in men, estrogen accounted for.70% of the total effect of sex steroids on boneresorption, whereas testosterone could account

for no more than 30% of the effect. Acutesex steroid deficiency clearly reduced thebone-formation markers (Fig. 2B), but theeffects of selective replacement differed forosteocalcin versus PINP. Reductions in osteo-calcin were prevented either by estrogen or tes-tosterone replacement, whereas only estrogen,but not testosterone, was able to prevent thereduction in PINP levels. Because osteocalcinis stored in the bone matrix, serum osteocalcinlevels may reflect not only bone formation butalso bone resorption (Ivaska et al. 2004). Incontrast, PINP is a pure bone-formation mark-er, so this evidence indicates that estrogen wasthe major regulator of bone formation in men.Note that these acute (3-week) effects of sexsteroid deficiency are, in fact, different fromthe chronic effects of sex steroid deficiency onbone-formation markers. In the latter situation,the “coupling” of bone resorption to bone for-mation (Khosla 2012) leads to increased bone

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Figure 2. Deconvoluting the effects of estrogen versus testosterone on bone metabolism in men. 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 amino-terminal extension peptide of type I collagen [PINP])in a group of elderly men (mean age 68 yr) made acutely hypogonadal and treated with an aromatase inhibitor(group A), treated with estrogen alone (group B), testosterone alone (group C), or both (group D). See text fordetails. Asterisks indicate significance for change from baseline: �P , 0.05; ��P , 0.01; ��P , 0.001. (FromFalahati-Nini et al. 2000; adapted, with permission, from the American Society for Clinical Investigation # 2000.)



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formation, but with a net gap between resorp-tion and formation and ongoing bone loss(Riggs et al. 2002).

The findings from this study were largelyconfirmed more recently by Finkelstein andcolleagues (Finkelstein et al. 2016) who useda somewhat different study design involvingpharmacological induction of hypogonadismin men followed by graded doses of testosteronereplacement with or without an aromataseinhibitor. Similar to our earlier study, changesin bone resorption and formation markerswere predominantly regulated by estrogen.Moreover, testosterone, in the absence of aro-matization to estrogen, was unable to preventdecreases in BMD at multiple sites. Collectively,these two and additional interventional studies(reviewed in Khosla 2015) using variations ofthe approaches used in the studies describedabove now provide compelling evidence inhumans that, in both women and men, estrogenis the dominant sex steroid regulating bone me-tabolism. However, testosterone likely does playan important role during growth and, directlyor indirectly (via the growth hormone/insulin-like growth factor [IGF] system) (Almeida et al.2017), contributes to the periosteal appositionthat results in larger bones in men as comparedwith women. We will revisit the findings fromthis human study in light of more recent find-ings in mice using cell-specific deletion of theandrogen receptor (AR), which may require areinterpretation of the human data.

Potential Mediators of Estrogen Action onBone in Humans

A limited number of interventional studies haveaddressed potential mechanisms or mediatorsof estrogen action on bone in vivo in humans.For example, based mainly on studies in ovari-ectomized rodents, a number of proinflamma-tory cytokines have been suggested as mediatorsof the effects of estrogen on bone resorption,with tumor necrosis factor (TNF)-a and inter-leukin (IL)-1b being perhaps the most consis-tent estrogen-regulated cytokines in the rodentmodels (for review, see Weitzmann and Pacifici2006). To address this issue in humans, we

designed a study using estrogen withdrawal fol-lowed by blockade of either TNF-a or IL-1baction in postmenopausal women (Charatcha-roenwitthaya et al. 2007). Specifically, postmen-opausal women were treated with estrogen for60 days followed by discontinuation of theestrogen for 3 weeks in the setting of eithersaline injections (control), treatment with aTNF-a blocker (etanercept), or an IL-1b block-er (anakinra). Because of the risk of infection,both blockers could not be used together inhumans. As shown in Figure 3, the increase inbone resorption in the control women was sig-nificantly blocked (by �50%) by the TNF-ablocker and to a lesser extent by the IL-1bblocker, showing that in humans TNF-a andperhaps IL-1bmediate the antiresorptive effectsof estrogen on bone. In this model, neither

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Figure 3. Effects of interleukin (IL)-1 or tumor ne-crosis factor (TNF) blockade on bone resorption inwomen following estrogen withdrawal. Proportionalchange (%) in serum C-telopeptide of type I collagen(CTx) in postmenopausal women treated for 60 dayswith transdermal estradiol, made acutely estrogendeficient, and treated with saline (control), an IL-1blocker (anakinra), or a TNF blocker (etanercept).(From Charatcharoenwitthaya et al. 2007; repro-duced, with permission, from John Wiley andSons # 2007.)



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blocker was able to prevent the decrease in boneformation following estrogen withdrawal, indi-cating that the maintenance of bone formationby estrogen was likely independent of these cy-tokines.

Another key factor regulating osteoclast de-velopment is receptor activator of nuclear factorkB (RANK) ligand (RANKL) produced by anumber of cells in the bone microenvironment,including osteocytes and osteoblast lineage cells(for review, see Kearns et al. 2008). Indeed,RANKL is both necessary and sufficient forosteoclast differentiation given permissive con-centrations of macrophage colony-stimulatingfactor (M-CSF), and it also enhances the activ-ity and prolongs the life span of osteoclasts bydecreasing apoptosis. To address the issue ofestrogen regulation of RANKL, we isolatedbone marrow mononuclear cells expressingRANKL on their surfaces using flow cytometryand the decoy RANKL receptor, osteoprote-gerin (OPG), as a probe (OPG-Fc-FITC) (Egh-bali-Fatourechi et al. 2003). The marrow cellswere characterized as osteoblast lineage, T cells,or B cells by using antibodies against bonealkaline phosphatase, CD3, and CD20, respec-tively, in premenopausal women, untreatedpostmenopausal women, or estrogen-treatedpostmenopausal women. We found that the

fluorescence intensity of OPG-Fc-FITC, anindex of the surface concentration of RANKLper cell, was increased in the estrogen-deficientpostmenopausal women as compared with thepremenopausal women by two- to threefold foreach of the cell populations, and these increaseswere reversed by estrogen treatment (Fig. 4).These data show that, directly or indirectly, es-trogen deficiency increases RANKL productionby a number of cell populations in the bonemicroenvironment, contributing to the increasein bone resorption. Notably, the increased boneresorption in postmenopausal women can nowbe entirely reversed clinically by treatment withdenosumab, a human monoclonal antibody toRANKL (Cummings et al. 2009).

There is also considerable evidence fromhuman studies that estrogen regulates the pro-duction of the proposed inhibitor of Wntsignaling, sclerostin (Drake and Khosla 2017).A number of studies have shown that treatmentof postmenopausal women with estrogen(Modder et al. 2011a,b; Fujita et al. 2014; Farret al. 2015) or with the selective ER modulator,raloxifene (Chung et al. 2012), reduces circulat-ing sclerostin levels using several different assaysfor sclerostin. Because there is some questionas to whether serum sclerostin levels reflectchanges in the bone microenvironment, we

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Figure 4. Effects of estrogen on cell surface receptor activator of nuclear factor kB ligand (RANKL) expression inpostmenopausal women. Changes in osteoprotegerin (OPG)-Fc-FITC fluorescence as an index of mean RANKLsurface concentration per cell are shown for premenopausal women (red bars), estrogen-deficient postmeno-pausal women (green bars), and estrogen-treated postmenopausal women (black bars). (A–D) The various cellpopulations as indicated. P-values by analysis of variance (ANOVA) are as indicated. ��P , 0.001 versus thepremenopausal women. (From Eghbali-Fatourechi et al. 2003; reproduced, with permission, from the AmericanSociety for Clinical Investigation # 2003.)



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recently examined bone sclerostin messengerRNA (mRNA) levels in needle bone biopsiesfrom either untreated or estrogen-treated post-menopausal women (Farr et al. 2015). Consis-tent with decreases in serum sclerostin levelsusing two different assays, bone sclerostinmRNA levels were also 41% and 48% lowerwhen assessed by RNAseq and QPCR, respec-tively, in the estrogen-treated versus controlwomen. Estrogen treatment also reduced bonemRNA levels of the sclerostin-related protein,sclerostin-containing protein 1 (SOSTDC1),which is also an inhibitor of Wnt- and BMP-signaling (Fujita et al. 2014). Given increasingevidence that sclerostin modulates not onlybone formation, but also bone resorption (Tuet al. 2015), these findings suggest that sclero-stin may be in the pathway of estrogen actionon bone, as shown further by the mouse modelsdiscussed later. Moreover, similar to the trans-lation of RANKL inhibition to a new therapeu-tic, sclerostin inhibition using a humanizedmonoclonal antibody, romosozumab (Cosmanet al. 2016), results in a profound increase inbone formation and reduction in bone resorp-tion in postmenopausal women, qualitativelysimilar to the pattern of changes in bone turn-over induced by estrogen treatment (Drake andKhosla 2017).

MOUSE MODELS

Deletion of ER or AR in Bone Cells

The effects of estrogen on bone are mediated bytwo related, but distinct, receptors, ERa andERb (Almeida et al. 2017). Although the roleof ERa in regulating bone metabolism in micehas been much more extensively studied thanthat of ERb, our understanding even of ERaaction on bone is still far from complete. Simsand colleagues (2002) initially studied micewith global deletion of ERa that had high cir-culating estrogen levels in females and increasedtestosterone levels in females and males. Dele-tion of ERa in either sex led to a decrease inbone turnover and increase in cancellous bonemass, but a decrease in cortical thickness. Thisindicated that, in cancellous bone, activation of

ERb by the high estrogen levels or the AR by thehigh testosterone levels in females was able tocompensate for loss of ERa in cancellous butnot cortical bone. Similarly, the high testoster-one levels in males were able to compensate forloss of ERa in cancellous but not cortical bone.Deletion of ERb did not alter sex steroid levelsin either sex, had no effect on bone in males,and led to a decrease in bone resorption andincrease in cancellous bone volume but not incortical thickness in females at 10 weeks of age.In contrast, a subsequent study by Windahl andcolleagues (1999) did find an increase in peri-osteal and endosteal circumference in femaleERb knockout mice. Thus, although the globalER-deletion models were confounded by alteredsex steroid levels in the ERa knockout mice,they did show that (1) loss of ERa compro-mised cortical bone thickness in both sexes,and (2) ERb did not regulate bone metabolismin males but could either compensate for loss ofERa in females, at least in the setting of elevatedestrogen levels, or when ERa was present, ERbappeared to antagonize ERa action on bone.

Similar to the global ERa knockout mice,male mice with global deletion of AR werefound to have significant alterations in circulat-ing sex steroid levels, specifically, reduced serumtestosterone levels (Yeh et al. 2002; Kawano et al.2003). Thus, although male AR knockout micehad reduced cancellous and cortical bone vol-ume because of increased bone resorption, it isunclear whether this was the result of loss of theAR in bone cells or the concomitant hypogo-nadism. In contrast to male AR knockout mice,female AR knockout mice did not have any skel-etal deficits.

Although these global ER and AR knockoutmice provided some insights, more conclusivedata on the roles of these receptors in bonemetabolism has come from studies using Cre/LoxP technology for cell-specific deletions.Although the original description by Ourslerand colleagues of the presence of ERs in avian(Oursler et al. 1991) and human (Oursler et al.1994) osteoclasts and effects of estrogen on di-rectly inhibiting osteoclast activity had beenmet with some skepticism, recent cell-specificERa deletions, in fact, point to the osteoclast as



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a key target of estrogen action in females, butnot males. Thus, ERa deletion either relativelyearly in the myeloid lineage using the LysM-Cre(Martin-Millan et al. 2010) or in the more dif-ferentiated osteoclast lineage using the Ctsk-Cre(Nakamura et al. 2007) led to reductions incancellous bone mass because of increasedosteoclast numbers and high levels of bone re-sorption, but only in female mice. In contrast,AR deletion in myeloid cells using the LysM-Credid not alter cancellous or cortical bone ineither sex (Ucer et al. 2015). Thus, ERa is crit-ical for directly regulating osteoclast numberand bone resorption in female but not malemice, and the AR does not appear to directlyregulate osteoclasts in either sex.

A number of studies have used osteoblastlineage Cre models to delete sex steroid recep-tors at various stages of osteoblast differentia-tion. Deletion of ERa in mesenchymal progen-itors (using Prx1-Cre) or in osteoprogenitors(using Osx-Cre) impaired Wnt/b-catenin sig-naling, reducing proliferation and differentia-tion of periosteal cells (Almeida et al. 2013).This signaling pathway was important for opti-mal cortical bone accrual in females with a tran-sient effect in males, but did not require thepresence of estrogen (i.e., was a ligand-indepen-dent effect of ERa). In contrast to cortical bone,deletion of ERa using either the Prx1-Cre orOsx-Cre had minimal or no effect in cancellousbone in either sex (Almeida et al. 2013).

ERa has also been deleted later in osteoblastdifferentiation using the Col1a1-Cre (Almeidaet al. 2013) but perhaps, surprisingly, neitherfemale nor male mice with ERa deletion usingthis Cre model showed deficits in bone mass ormicroarchitecture at 4, 8, 12, or 26 weeks of age.Interestingly, the ERaf/f;Col1a1-cre mice didshow an increase in osteoblast apoptosis incancellous bone, but this did not translate intostructural alterations in bone. In contrast tothese findings using the Col1a1-Cre, studiesusing Cre models activated even later in osteo-blast differentiation (i.e., in the late osteoblast/osteocyte) have generally found a reduction inbone mass in both sexes associated with lowbone-formation rates, although the data aresomewhat inconsistent. Thus, two studies used

the osteocalcin (Ocn)-Cre and found deficitsin cancellous and in cortical bone associatedwith reduced cancellous bone formation ratesin female mice (Maatta et al. 2013; Melvilleet al. 2014). Although these deficits were presentin female mice at 3–4 months of age, they ap-peared to take longer to develop in male miceand only became evident at 6 months of age.

Studies using the Dmp1-Cre, which over-laps with the Ocn-Cre but is expressed perhapssomewhat later in osteoblast differentiation,have yielded somewhat conflicting results.Thus, Windahl et al. (2013) reported thatmale but not female mice with ERa deletionusing the Dmp1-Cre had reduced cancellousbone mass associated with decreased bone for-mation. In contrast, Kondoh et al. (2014) foundthe opposite—reduced cancellous bone massand decreased bone formation in female butnot male mice. Cortical bone was unaffectedin either sex in both studies. The reasons forthese discrepant findings are unclear, but thecollective findings using the Ocn- and Dmp1-Cre models do indicate that ERa signaling islikely important in mature osteoblasts/osteo-cytes and that ERa regulates bone formation,at least in part through regulating osteoblastapoptosis.

Cell-specific deletion of ERb either in osteo-progenitor cells (Prx1-Cre) or in more matureosteoblasts (Col1a1-Cre) has been shown tohave the opposite effect to ERa deletion, name-ly, an increase in cancellous bone mass butwithout an alteration in cortical bone mass infemale mice (Nicks et al. 2016). This compart-ment-specific effect of ERb deletion (i.e., anincrease in cancellous but not cortical bonemass) is of interest, as previous studies inhuman (Bord et al. 2001) and mouse (Modderet al. 2004) bone have found that both ERa andERb are expressed in cancellous bone, whereascortical bone mainly contains ERa. In addition,ex vivo studies found an increase in the ratio ofcolony-forming unit osteoblasts (CFU-OBs) toCFU fibroblasts (CFU-Fs) in the ERb-deletedmice, indicating increased differentiation ofosteoblast precursor cells into osteoblasts inthe absence of ERb. Consistent with findingsin reproductive tissues (Hall and McDonnell



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1999), ERb was shown to antagonize ERaaction in osteoblastic cells (Nicks et al. 2016).Thus, ERb likely plays an antagonistic role toERa in bone, and this effect is predominant incancellous bone. This point will be consideredfurther below in attempting to understand thecompartment-specific effects of sex steroids infemales and males.

In contrast to the absence of skeletal effectsof AR deletion in myeloid/osteoclast lineagecells, deletion of the AR in osteoprogenitor cells(Prx1-Cre) resulted in osteopenia and increasedbone resorption in cancellous, but not in corti-cal, bone in male mice, with female mice havinga much milder but qualitatively similar pheno-type (Ucer et al. 2015). Very similar findingswere reported using the Col1a1-, Ocn-, andDmp1-Cre mice crossed with floxed AR mice(Notini et al. 2007; Chiang et al. 2009; Sinne-sael et al. 2012), establishing the consistency ofthis observation across osteoblast lineage Cremodels. Moreover, these unequivocal effects ofAR signaling in cancellous bone in male miceappear to be at odds with the clear evidence fromhuman studies noted earlier that estrogen is thedominant regulator of bone metabolism in men.This apparent discrepancy between the mouseand human data will be considered below.

Table 1 attempts to summarize the now ex-tensive body of work from multiple laboratoriesregarding global, osteoblast-, or osteoclast-lin-eage deletion of ERa, ERb, or AR. Keeping inmind that not all of the data are consistent

across laboratories, the table presents the overallpicture that is emerging. Thus, although globalERa deletion is confounded by profound alter-ations in circulating sex steroid levels, bothfemale and male ERa knockout mice do havereductions in cortical bone mass. The most un-equivocal findings are the following: (1) ERadeletion in osteoclasts leads to reduced cancel-lous (but not cortical) bone mass in females,but not males; (2) AR deletion in osteoblastlineage cells leads to reduced cancellous (butnot cortical) bone mass in males, with a milderphenotype in females; and (3) ERb deletionin osteoblast lineage cells leads to increasedcancellous (but not cortical) bone mass in fe-males. ERa deletion in osteoblast lineage cellshas generally resulted in reductions in cancel-lous and perhaps cortical bone mass in femalesand cancellous bone mass in males, but thesefindings have been somewhat variable acrosslaboratories. However, an important caveat toall of these studies is that the receptor deletion,although cell-specific, has been present sinceconception. As such, the skeletal findings reflectlargely developmental effects of these receptorsrather than their role in the adult skeleton.Further studies using both cell- and temporal-specific deletion of these receptors using induc-ible models are needed to address this limitationand further clarify the role of sex steroid recep-tors on the adult murine skeleton. In addition,for each of these receptor deletions, the severityof the published phenotypes depends on the

Table 1. Summary of effects of deletion of sex steroid receptors, either globally or in specific cells, on bone mass

Receptor deleted

Global

Osteoblast lineage/

osteocyte Myeloid/osteoclast

Cancellous Cortical Cancellous Cortical Cancellous Cortical

Female ERa � � (�) (�) � $ERb � $ � $ NT NTAR $ $ � $ $ $

Male ERa � � (�) $ $ $ERb $ $ NT NT NT NTAR � � � $ $ $

Parentheses surrounding arrows indicate somewhat conflicting data, with the overall evidence pointing to the direction

noted.

NT, Not tested.



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efficiency of deletion of the gene as well as thespecificity of the deletion in the particular celltype, which is not always clearly assessed in theoriginal papers.

ERa Deletion in the Central Nervous System

As shown by the above studies, ERa clearly hasdirect effects on bone, acting to preserve orenhance bone mass. In contrast, several studiesindicate that this positive peripheral effect ofERa may be counterbalanced by a negativecentral effect. Thus, deletion of ERa in the cen-tral nervous system (CNS) using the nestin-Creresulted in an increase in cancellous and corticalbone mass in female mice associated with in-creased bone formation (Ohlsson et al. 2012).The CNS ERa knockout mice had normal sexsteroid, serotonin, and catecholamine levels,but did have increased serum leptin levels.Generally similar findings were noted in asubsequent study using POMC-Cre mice, inwhich the ERa deletion was more specific forhypothalamic neurons (Farman et al. 2016).Although this negative central effect of estrogensignaling on bone is of interest, the net effectof estrogen deficiency in mice and humans isbone loss, arguing that the peripheral effectsof ERa signaling dominate over these negativecentral effects.

Role of T and B Cells

A series of studies, principally in mice, haveprovided substantial evidence for an importantrole of T cells in mediating ovariectomy-in-duced bone loss. The fundamental observationis that ovariectomy leads to an increase in TNF-producing T cells, which in turn results in anincrease in RANKL-induced osteoclast forma-tion (for review, see Pacifici 2012). A number ofmechanisms have been proposed for the expan-sion of TNF-producing T cells, includingincreased antigen presentation, stimulation ofIL-7 and interferon (IFN)-g production, anddown-regulation of transforming growth factor(TGF)-b production (Pacifici 2012). ActivatedT cells also increase RANKL and M-CSF pro-duction by bone marrow stromal cells through

CD40L and DLK1/FA-1 (Pacifici 2012). Afurther intriguing aspect of this work hasbeen the recent evidence linking sex steroiddeficiency in mice to gut microbiota (Li et al.2016). Specifically, sex steroid deficiency ap-pears to lead to increased gut permeability, anexpansion of Th17-activated T cells, and subse-quent up-regulation of osteoclastogenic cyto-kines, including TNF-a, RANKL, and IL-17.Remarkably, germ-free mice lacking an intesti-nal microbiome are resistant to sex steroiddeficiency-induced cancellous bone loss. Thisarea is not without some controversy, however,as various mouse strains lacking T cells do losecancellous bone following ovariectomy (Leeet al. 2006). In addition, mice lacking RANKLspecifically in T cells lose bone similar to controlmice following ovariectomy (Nakashima et al.2011; Onal et al. 2012).

There is also evidence linking B cells toovariectomy-induced bone loss in mice. Specif-ically, deletion of RANKL from B cells preventscancellous bone loss and increased bone resorp-tion following ovariectomy in mice (Onal et al.2012). Ovariectomy increases the number ofB cells (Masuzawa et al. 1994), although, inhumans, estrogen deficiency may also increaseRANKL expression per cell on B cells (Eghbali-Fatourechi et al. 2003). However, the precisemechanisms by which estrogens or androgenscontrol B-cell numbers remain unclear at thispoint.

Potential Mediators of Estrogen Actionfrom Mouse Models Relative to Findingsin Humans

As noted earlier, there is relative consistencybetween the mouse data showing an importantrole for TNF-a in mediating ovariectomy-induced bone loss in mice (Weitzmann andPacifici 2006) and human studies using theTNF-a blocker (etanercept) (Charatcharoen-witthaya et al. 2007). Estrogen deficiency alsoleads (directly or indirectly) to increasedRANKL production by a number of cells, oran expansion of cells expressing RANKL, inthe bone microenvironment in mouse models(Pacifici 2012), again generally consistent with



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human studies showing increased RANKL ex-pression on bone marrow stromal cells, T cells,and B cells in estrogen deficient as comparedwith estrogen-sufficient women (Eghbali-Fa-tourechi et al. 2003). The human studies show-ing regulation of circulating sclerostin and boneSOST mRNA levels by estrogen (Modder et al.2011a,b; Fujita et al. 2014; Farr et al. 2015) arealso consistent with studies in mice with Lrp5mutations rendering them resistant to sclerostin(Niziolek et al. 2015). These mice have absent ormarkedly attenuated bone loss following ovari-ectomy, indicating a potential role for sclerostinin mediating estrogen-deficiency bone loss. Thesuppression of SOSTDC1 mRNA levels in bonein postmenopausal women treated with estro-gen (Fujita et al. 2014) is also consistent with theobservation that mice with osteocyte-specificdeletion of ERa have increased bone sostdc1mRNA levels (Kondoh et al. 2014), althougha causative role for sostdc1 in facilitating ormediating estrogen deficiency–induced boneloss remains to be established. ERa deletionin mesenchymal/stromal cells has also beenshown to increase the expression of SDF1 andMMP13 (Ucer et al. 2016), which may play arole in mediating increased endocortical boneresorption following estrogen deficiency. How-ever, these findings have not yet been validatedin human studies.

DISCREPANCIES BETWEEN THE HUMANAND MOUSE STUDIES AND POSSIBLERESOLUTIONS

Although there generally are consistencies be-tween the human and mouse data regarding sexsteroid action on bone, and the mouse studieshave been instrumental in defining the specificroles of ERa, ERb, and AR in bone metabolism,there is a fundamental discrepancy between thehuman and mouse models that needs to be re-solved. As noted earlier, the collective evidencefrom both the observational and interventionalhuman studies is overwhelming that estrogenplays a much more significant role in regulatingbone metabolism in men than testosterone (forreview, see Khosla et al. 2008). In contrast, fourseparate studies in mice, discussed above, have

found that AR deletion in osteoblast lineagecells in male mice leads to reduced cancellousbone volume and cancellous number associatedwith increased osteoclast numbers (Notini et al.2007; Chiang et al. 2009; Sinnesael et al. 2012;Ucer et al. 2015). Moreover, neither AR nor ERadeletion has any effect on cortical bone in malemice.

One possibility is that mice may be funda-mentally different from humans, at least interms of sex steroid regulation of male bone.This may be a result of the fact that aromataseexpression in rodents is predominantly in thebrain and gonads, whereas humans have exten-sive aromatase expression across tissues (Simp-son et al. 1997). On the other hand, it may bethat the original interpretation of the humandata (including by our group) was incomplete,particularly in light of the subsequent findingsin mice. Thus, in our original human studyusing depletion of sex steroids and selectivereplacement with estrogen or testosterone (Fa-lahati-Nini et al. 2000), we did observe a smallincrease in NTx in the 2T, þE group (Fig. 2A),which represented �30% of the NTx increaseobserved after complete sex steroid deficiency.In light of the mouse findings, this increase,which was not suppressible by estrogen, likelyrepresents the loss of the direct effects of testos-terone (without aromatization to estrogen) onbone resorption in cancellous bone. In this in-terpretation, the bulk of the effects of estrogenin human males may well be in cortical bone,which is fundamentally different in humansversus mice. Specifically, humans have extensiveintracortical, osteonal remodeling that is absentin mice (Jikla 2013). Thus, consistent with thefindings in the AR deletion mouse models, tes-tosterone (in the absence of aromatization toestrogen) likely does regulate cancellous boneresorption in humans, but the regulation ofcortical bone remodeling by estrogen is muchmore evident in human versus mouse malesbecause of the fundamental differences incortical bone between the species. Moreover,because �80% of the skeleton is cortical bone(Bonnick 1998), estrogen inevitably emerges asthe more dominant regulator of bone metabo-lism in human males.



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Based on these considerations, Figure 5 at-tempts to synthesize the findings from the hu-man and mouse models and presents a workinghypothesis for the compartment-specific effectsor estrogens versus androgens on bone in fe-males and males. As described earlier, the majoreffect of castration in either sex is an increasein bone remodeling, with bone resorption out-stripping bone formation, leading to bone loss.Thus, sex steroids collectively restrain the acti-vation of bone remodeling, likely througheffects on osteocytes, although this needs addi-tional experimental validation using inducibledeletion of sex steroid receptors in adult mice.As noted earlier, the distribution of ERa versusERb appears to be quite different in cancellousand cortical bone, with both receptors ex-pressed in cancellous bone and ERa predomi-nating in cortical bone. Because ERb antago-nizes ERa in bone (Nicks et al. 2016) andother tissues (Hall and McDonnell 1999), aplausible hypothesis that is supported byhuman (Khosla et al. 2011) and mouse (Syedet al. 2010) data is that it would take higherlevels of estrogen to suppress bone remodelingin cancellous as compared with cortical bone. Infemales (Fig. 5A), estrogen levels are sufficientlyhigh so that estrogen consistently suppressesbone remodeling in both cortical and cancel-lous bone, with testosterone playing a minimalrole in cancellous bone and no discernablerole in cortical bone. Males, however, havemuch lower estrogen levels, which are able tosuppress cortical bone remodeling and perhapshave some effects in cancellous bone, but malesrequire substantial androgen action in cancel-

lous bone to adequately restrain bone remodel-ing and prevent bone loss in that compartmentas adults (Fig. 5B). Although lacking experi-mental proof in all aspects, this formulationis nonetheless consistent with the availabledata discussed in this perspective and does rec-oncile the seemingly disparate findings fromhumans versus mice, keeping in mind the fun-damental difference in intracortical remodelingbetween species.

SUMMARY AND CONCLUSIONS

Since the original description by Fuller Albrightof the effects of estrogen deficiency on bonein women (Albright 1940), there has been enor-mous progress in our understanding of sexsteroid regulation of the skeleton. Studies inhumans and mice have generally been concor-dant and helped define the target cells, the rolesof ERa, ERb, and AR, and potential mediatorsof estrogen action on bone, albeit with less in-formation on downstream targets of androgenaction. The importance of estrogen action onbone is reflected by the fact that it is a key reg-ulator of bone metabolism not only in womenbut also in men. Moreover, two of the new drugsdeveloped in recent years for the treatment ofosteoporosis, denosumab and romozosumab,target RANKL and sclerostin, respectively—molecules that in humans and in mice havebeen implicated in mediating, at least in part,estrogen action on bone. Thus, continuing tobetter define the mechanisms of sex steroid ac-tion on bone will not only provide fundamentalknowledge regarding the regulation of bone

FemalesA B

Cancellous boneERα, ERβ

AR

Cortical boneERα>>ERβ

AR

HighE2

LowT

Cancellous boneERα, ERβ

AR

Cortical boneERα>>ERβ

AR

LowE2

HighT

Males

Figure 5. Working model to explain the differential effects of estrogen versus testosterone on cancellous andcortical bone. Please see text for further details. ER, Estrogen receptor; AR, androgen receptor.



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metabolism, but likely also identify additionaltherapeutic targets to treat osteoporosis.

ACKNOWLEDGMENTS

This work is supported by National Institutesof Health (NIH) Grants P01 AG004875,AG048792, AR027065, and AR068275.

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14, 20172018; doi: 10.1101/cshperspect.a031211 originally published online JulyCold Spring Harb Perspect Med

Sundeep Khosla and David G. Monroe Regulation of Bone Metabolism by Sex Steroids

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Regulation of Bone Metabolism by Sex Steroidsperspectivesinmedicine.cshlp.org/content/8/1/a031211... · 2017-12-20 · Regulation of Bone Metabolism by Sex Steroids Sundeep Khosla