Hormonal Effects on Bone Cells

8/18/2019 Hormonal Effects on Bone Cells

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C H A P T E R

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Hormonal Effects on Bone CellsTeresita Bellido1 and Kathleen M. Hill Gallant2

1Roudebush Veterans Administration Medical Center, Indianapolis, Indiana, USA 2Department of Anatomy and Cell

Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA

INTRODUCTION: DIRECT VERSUSINDIRECT EFFECTS OF HORMONES ON

BONE CELLSSystemic hormones can affect bone either directly or

indirectly. Direct action occurs through receptorsexpressed in bone cells. Indirect action occurs when ahormone modulates mineral homeostasis through regu-lation of calcium and phosphate absorption by the intes-tine and excretion or reabsorption by the kidney. Thegoal of this chapter is to discuss the current knowledgeabout the direct effects of hormones on the skeleton.

PARATHYROID HORMONE

Parathyroid hormone (PTH) is a peptide hormonethat controls the minute-to-minute level of ionized cal-cium in the circulation and extracellular fluids. PTH issecreted by the chief cells of the parathyroid gland inresponse to low levels of calcium in the blood. Thetwo main target tissues of PTH are bone and kidney.By binding to receptors in cells of these tissues, PTHinduces responses leading to an increase in bloodcalcium concentrations. This increase in circulatingcalcium, in turn, feeds back on the parathyroid glandto reduce PTH secretion.

Actions of Parathyroid Hormone on Bone

The primary effect of PTH on the skeleton is toinduce bone resorption with the goal of liberating cal-cium from the mineralized matrix and increasing itsconcentration in the blood and extracellular fluids.

PTH has profound effects on the skeleton at thetissue level. Elevated circulating levels of the hormone

can generate both catabolic and anabolic effects on bone,depending on the temporal profile of its increase.

Continuous (or chronic) elevations in PTH, as in primaryor secondary hyperparathyroidism, increase the rate of bone remodeling, and can result in loss of bone. In con-trast, intermittent increases of PTH in the blood, asachieved by daily injections of the pharmaceutical agentteriparatide [recombinant human PTH; rhPTH(134)],results in bone gain.

The high bone remodeling rates and bone loss result-ing from chronic PTH elevation are associated withexcessive production and activity of both osteoclasts andosteoblasts. The enhancement of osteoclast activity out-paces that of osteoblasts and thus results in a negative

basic multicellular unit (BMU) balance (see Chapter 4,

Fig. 4.11). Conversely, the primary effect of intermittentPTH elevation is a rapid increase in the number andactivity of osteoblasts and in bone formation, leading tonet bone gain. The mechanism of this anabolic effect isattributed to the ability of PTH to promote proliferationof osteoblast precursors, inhibit osteoblast apoptosis,reactivate lining cells to become matrix synthesizingosteoblasts, or a combination of these effects (see below).In humans, intermittent PTH administration stimulates

bone formation by increasing the bone remodeling rateand the amount of bone formed by each BMU in a pro-cess named remodeling-based formation. PTH also stimu-lates bone formation not coupled to prior resorption,

referred to as modeling-based formation. The latter mecha-nism appears to be more evident in rodents.

Parathyroid Hormone Receptors andDownstream Signaling

PTH binds with high affinity to the parathyroidhormone/parathyroid hormone-related peptide

299Basic and Applied Bone Biology.

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receptor (PTH1-R), which belongs to the family of Gprotein-coupled receptors, and also binds PTH-related peptide (PTHrP). In bone, only cells of themesenchymal/osteoblastic lineage express PTH1-R.Therefore, the effects of PTH in bone are mediated

by osteoblasts, even though a major function of thehormone is to increase bone resorption via osteo-clasts. As with other hormones whose receptors are

coupled to G proteins, PTH activates downstreamsignaling of Gα (cAMP) and Gβγ proteins [phosphoi-nositide 3-kinase (PI3-K) and phospholipase C(PLC)]. It is recognized, however, that the majoreffects of PTH in bone are downstream of the cAMPsignaling pathway.

Effects of Parathyroid Hormone on Osteoblastsand Bone Formation

A major effect of PTH is to increase osteoblast numberand enhance the rate of bone formation. Different

mechanisms might operate depending on both the modeof elevation and the bone envelope (Fig. 15.1). Studies inanimals suggest that intermittent and chronic PTH eleva-tions increase osteoblast number by distinct mechan-isms. The anabolic effect of intermittent PTH incancellous bone can be accounted for by attenuation of osteoblast apoptosis, whereas the increase in bone for-mation on the periosteal surface of cortical bone appearsto result from reactivation of lining cells to become activeosteoblasts. In contrast, chronic elevation of PTH has noeffect on osteoblast survival. Its osteoblastogenic actionresults from direct actions of the hormone on osteocytesinhibiting the expression of the SOST gene and its prod-

uct sclerostin, an inhibitor of bone formation. Sclerostindownregulation is responsible for the increase in boneformation in cancellous as well as in both periosteal andendocortical surfaces of cortical bone.

Inhibition of Osteoblast Apoptosis byParathyroid Hormone

Increased cell survival is a major contributor to theincrease in osteoblast number caused by intermittentPTH administration (Fig. 15.1). Daily injections of PTHcause a dose-dependent increase in bone mineral den-sity (BMD) associated with a reduction in osteoblast

apoptosis, and increased osteoblast number, boneformation rate, and the amount of cancellous bone. Invitro, PTH or PTHrP inhibits apoptosis in cultured rat,murine, and human osteoblastic cells. This occurs viacAMP-activated protein kinase A (PKA), inactivation of the proapoptotic protein Bad, as well as increased tran-scription of survival genes like Bcl-2. The increased syn-thesis of survival genes requires the cAMP-responsiveelement-binding protein (CREB) and RUNX2. Thesefindings suggest that the decreased osteoblast apoptosisin response to intermittent PTH is probably due to short

bursts of survival signaling in osteoblasts. Besides anti-apoptotic effects downstream of cAMP activation, sur-

vival of osteoblasts induced by PTH might requiresignaling activated by locally produced factors, such asfibroblast growth factor 2 (FGF-2), insulin-like growthfactor (IGF-I), and Wnts.

Downregulation of Sclerostin by ParathyroidHormone

Evidence that PTH inhibits the expression of theosteocyte-derived inhibitor of bone formation, scleros-tin, provided the basis for a novel mechanism bywhich the hormone could affect skeletal homeostasis

through effects on osteocytic gene expression anddemonstrates that osteocytes are crucial target cells of PTH in bone (Fig. 15.1). Continuous treatment withPTH markedly suppresses Sost mRNA and sclerostin

SclerostinPTH

-

OsteocytePTH

+

PTH

+

Lining cell

Mesenchymal

stem cell

Mature

osteoblastPTH

–

–

Apoptotic

osteoblastBone formation

Wnt signaling

FIGURE 15.1 Parathyroid hormone stimu-lates bone formation by regulating osteoblast

generation and life span. Parathyroid hormone(PTH) promotes survival of mature osteoblasts,thus prolonging their matrix synthesizing func-tion. In osteocytes, PTH inhibits the expressionof sclerostin, an inhibitor of bone formation,potentiating the stimulatory effect of Wnt signal-ing on osteoblast differentiation. PTH may alsoreactivate quiescent lining cells to becomematrix synthesizing osteoblasts. 1, stimulation;2, inhibition.

300 15. HORMONAL EFFECTS ON BONE CELLS

4. HORMONAL AND METABOLIC EFFECTS ON BONE


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protein expression in rodent models. This effect isreproduced in vitro in osteocytic and osteoblastic celllines and in primary cultures of calvaria cells contain-ing osteocytes, demonstrating that it results from adirect effect of PTH on its receptor expressed by osteo-cytes, rather than arising from hormonal actions onother bone cells or indirectly through other tissues.Intermittent PTH administration also reduces Sost

expression, but to a lesser extent and only transientlyafter each daily injection. Thus, sustained downregula-tion of sclerostin appears not to be required for boneanabolism induced by intermittent PTH; however, it islikely that repetitive reductions in sclerostin could bepart of the increase in bone formation induced by thehormone. These findings have been independentlyconfirmed using several animal models and also vali-dated in humans.

PTH exerts its inhibitory effect on sclerostin expres-sion downstream of PTH1-R-cAMP pathway. This isdemonstrated by the fact that PTHrP, the other ligandof this receptor, and stable analogs of cAMP, mimicthe effects of PTH on Sost. However, Sost downregula-tion appears not to depend on transcription factors of the CREB family. Instead, transcription factors of themyocyte-specific enhancer factor (MEF2) family medi-ate the effect of PTH on Sost expression. Nevertheless,the exact molecular mechanism of this regulationremains unknown.

Expression of a constitutively active PTH1-R inosteocytes in transgenic mice is sufficient to downre-gulate Sost and reduce sclerostin levels in vivo. This isassociated with increased Wnt activation, marked stim-ulation of bone formation and increases in bone mass.

Bone formation and bone mass are reversed to wild-type levels in double transgenic mice also expressingSost in osteocytes, demonstrating that Sost downregu-lation is needed to induce bone anabolism by PTH1-Rsignaling in osteocytes.

Reactivation of Lining Cells by ParathyroidHormone

Other mechanisms besides downregulation of Sostexpression and osteoblast survival are likely to contrib-ute to the profound skeletal effects of PTH on bone

formation. One of these additional mechanisms is theconversion of inactive lining cells that cover the quies-cent surface of bone into matrix-producing osteoblasts(Fig. 15.1). This mechanism was suggested by indirectstudies showing that PTH increases osteoblast numberon bone surfaces concomitantly with a decrease inlining cell number, without detectable changes in cellproliferation. A more recent lineage-tracing studyshowed that PTH is able to convert lining cells into

osteoblasts on periosteal bone surfaces, adding supportto this hypothesis.

Effects of Parathyroid Hormone onOsteoclastogenesis and Bone Resorption

PTH promotes osteoclast formation by upregulating

RANK ligand [tumor necrosis factor ligand superfamilymember 11/receptor activator of the NF-κB ligand(RANKL)], encoded by TNFRSF11B], downregulatingosteoprotegerin/tumor necrosis factor receptor super-family member 11B (OPG), and thus increasing theRANKL:OPG ratio (Fig. 15.2). This has been demon-strated by several in vitro studies with cultured stromal/osteoblastic cells that support osteoclast formation. Inaddition, mice lacking either PTH or the enhancer regu-lated by PTH in the Tnfrsf11b gene [distal control region(DCR)] exhibit low RANKL expression and low boneremodeling. Moreover, increased RANKL expressioninduced by endogenous elevation of PTH or by lactation

mediated by PTHrP is abolished in DCR2/2 mice.It is established that the pro-osteoclastogenic action of

PTH is mediated by cells of the osteoblastic lineage.OPG is expressed by osteoblasts and osteocytes; how-ever, the differentiation stage of the PTH target cell thatsupports osteoclast formation has remained obscure.Recent evidence shows that osteocytes express RANKL

Stromal/osteoblastic

cellsOsteocytesOsteoblasts

PTH PTH

M-CSF, RANKL OPG

PTH PTH

– +

Hematopoietic osteoclast

precursor Mature

osteoclast

Bone resorption

FIGURE 15.2 Parathyroid hormone stimulates bone resorptionby regulating the expression of pro- and anti-osteoclastogenic cyto-

kines in cells of the osteoblastic lineage. Osteoclast differentiationfrom hematopoietic precursors of osteoclasts is stimulated by theRANK ligand [tumor necrosis factor ligand superfamily member 11/receptor activator of the NF-κB ligand (RANKL)] and macrophagecolony-stimulating factor 1 (M-CSF) and inhibited by osteoprotegerin(OPG). Parathyroid hormone (PTH), acting on receptors expressed incells of the osteoblastic lineage, increases osteoclast production and

bone resorption by increasing RANKL and inhibiting OPG. 1, stimu-lation; 2, inhibition.

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and that deletion of RANKL from osteocytes leads toosteopetrosis. Moreover, RANKL expression, osteoclastnumber, and bone resorption are elevated in transgenicmice with constitutive activation of the PTH1-R in osteo-cytes. These findings raise the possibility that at leastpart of the effects of PTH on osteoclast differentiationand resorption are due to osteocytic RANKL regulation.

SEX STEROIDS

In the 1940s, Fuller Albright made the association between women’s loss of estrogen at menopause and bone loss. For decades, this association was believed to be indirect, until the discovery in the late 1980s thatestrogens bind directly to bone cells, indicating a directeffect of estrogen on the skeleton. In men, the gradualreduction in androgen secretion with aging is associ-ated with bone loss. Some of the effects of androgensare due to their conversion to estrogen. However, bone

cells express receptors that specifically bind androgensand mediate their biological effects independently of estrogens. This section addresses the general and sex-specific effects of the main sex steroid hormones affect-ing skeletal tissue: androgens and estrogens.

Sex Steroid Production

Sex steroid hormone synthesis begins by hydrolysisof cholesterol esters and uptake of cholesterol by themitochondria of target tissue cells. Cholesterol ismetabolized to pregnenolone, which is further metabo-lized to produce all sex steroid hormones. Estrogens

are sex steroids secreted by the ovaries in women andto a small extent by the testes in men. Over 80% of estrogen in men is produced through peripheral

conversion of androgens to estrogens by cytochromeP450 aromatase. Adipose tissue is the main tissue forestrogen production in men and for extraovarian estro-gen production in women.

Androgens are sex steroids secreted by the testes inmen, the ovaries in women, and the adrenal glands in

both men and women. Testosterone, the main andro-gen in men, is secreted primarily by the testes (approx-

imately 95% of total testosterone). In women, onlyabout 25% of testosterone comes from the ovaries;another 25% comes from the adrenal glands, but half of total testosterone in women comes from conversionof other sex steroids, such as dehydroepiandrosterone(DHEA) and androstenedione, by peripheral tissuessuch as adipose tissue.

Most testosterone is bound to proteins in the circu-lation. Approximately half is bound with high affinityto steroid hormone-binding globulin, with the otherhalf bound with low affinity to albumin. Only 12%of testosterone is free (unbound) in the circulation.Bioavailable testosterone refers to both free testoster-one and albumin-bound testosterone. Free testosteronediffuses passively through cell membranes and bindsto the androgen receptor. Testosterone can be metabo-lized in peripheral tissues to the potent androgen,dihydrotestosterone by 5-alpha-reductase, or to 17β-estradiol by cytochrome P450 aromatase.

Sex Steroid Receptor Signaling

Sex steroid signaling occurs through genotropicand nongenotropic signaling pathways (Fig. 15.3).Genotropic signaling occurs when the sex steroid ligands

bind to the sex steroid receptors, which then dimerizeand translocate to the nucleus to initiate gene transcrip-tion. Dimerized sex steroid receptors can bind directly to

Genotropic mechanisms

Receptor-mediated

rapid kinase activationDirect receptor-DNA interaction

ERaCoActSH2 / SH3E

E

ERa ERa

EREC3 Shc

PSrc

PP

E

Receptor-transcription factor interaction MEKP

p50 p65ERKs

E E

ERa ERaPosttranslational changes

and gene expression

IL-6Osteoblast/osteocyte

survival

NF-kB

Nongenotropic mechanism FIGURE 15.3 Signaling pathways activated bysex steroids. Estrogens (ERs; depicted in the figure)and androgens (not shown) activate genotropic andnongenotropic pathways. CoAct, coactivator; E, estra-diol. See text for details.




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response elements in promoters of the target genes[e.g. estrogen response elements (EREs), for estrogens].Alternatively, receptor monomers can directly interactwith transcription factors, and these complexes subse-quently bind to promoters in the target genes throughresponse elements for the particular transcription factors.Major transcription factors that participate in sex steroidsignaling include nuclear factor-kappa-B (NF-κB) and

activator protein 1 (AP-1).Sex steroids also activate rapid, kinase-mediated sig-

naling by binding to membrane-bound receptors. Rapidsignaling is initiated by binding of the ligand to thereceptor at the cell membrane. Signaling is amplifiedthrough the interaction of receptors with scaffolding pro-teins and culminates with activation of kinases, includ-ing Akt, proto-oncogene tyrosine-protein kinase Src,extracellular signal-regulated kinases (ERKs), PI3-K,PKA, and protein kinase C (PKC). This mechanism has

been termed nongenotropic because it does not involvedirect binding of the receptor to DNA. However, it isimportant to note that kinase signaling leads not only toposttranslational changes in proteins (such as phosphor-ylation) but also to transcriptional changes that involvealterations of gene expression mediated by kinase-activated transcription factors.

Sex Steroids during Growth

At puberty, boys and girls experience a period of rapid height gain followed by a period of rapid bone

mineral accrual. Girls experience these growth spurtson average a year and a half before boys, but boysachieve higher peak height velocity and peak bone min-eral velocity. Subsequently, boys are taller and havegreater bone mass than girls by the end of puberty, andultimately have higher peak bone mass in adulthood.

The sexual dimorphism of the skeleton duringgrowth is attributed to a general stimulatory effect of

androgens and inhibitory effect of estrogens on bonegrowth. Androgens appear to be stimulatory forperiosteal bone expansion, which is greater in boysthan girls during puberty and throughout the yearsof peak bone mass acquisition. Androgen deficiencyin males results in a reduction in periosteal boneexpansion. Conversely, estrogens are inhibitory of periosteal expansion, as estrogen-deficient femaleshave a drastic increase in periosteal bone expansion.On the endocortical surface, estrogens promote andandrogens suppress bone formation during growth.As a result, girls at puberty have cortical thickeningfrom endosteal contraction with little periostealexpansion, whereas boys have cortical thickeningmostly from periosteal expansion being greater thanendosteal expansion (Fig. 15.4). Estrogen signalingthrough ERβ appears to be responsible for the effectsof estrogen on the periosteal and endosteal surfaces.Thus, female ERβ knockout mice have bones thatresemble wild-type males, with greater periostealand endosteal circumferences and greater cross-sectional diameter.

Sex hormone effects on longitudinal growthEarly Puberty

Late pubertyHypothalamus +

pituitary

ERα AR

EGH-IGF-1

AxisERα

T

E

Longitudinal

growth

Epiphyseal

closure

>

Sex hormone effects on bone surfaces

ERβ Corticalthickening

with endostealcontraction

ERαE

+

–

Growth

T AR+ Corticalthickening

with periosteal

expansion

FIGURE 15.4 Concept model of theeffects of sex hormones during growth.Early in puberty, low levels of estrogenand testosterone stimulate longitudinal

bone growth. In both sexes late in puberty,estrogen stimulates epiphyseal closure.Estrogen is stimulatory to bone formationat the endosteal surface and inhibitory atthe periosteal surface of bone, whereas tes-tosterone is stimulatory at the periostealsurface. AR, androgen receptor; GH,somatotropin/growth hormone; E, estro-gen; ERα/β, estrogen receptor α/β; IGF-I,insulin-like growth factor I; T, testosterone.See text for details.

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At the beginning of puberty, both estrogen and tes-tosterone activate the somatotropin/growth hormone(GH)-IGF-I axis to stimulate longitudinal bone growth.The effects of estrogen during growth are dependenton the stage of development. Early in puberty, estro-gen (at relatively low levels in girls compared withlater puberty) signaling through ERα in the hypothala-mus and pituitary is necessary for GH secretion, which

acts directly and indirectly through IGF-I to increaselongitudinal bone growth by stimulating proliferationof growth plate cartilage. At the end of puberty, estro-gen levels are high and act directly through ERα sig-naling in growth plate chondrocytes to slow and thencease longitudinal bone growth. Estrogen signalingthrough ERα is responsible for epiphyseal closure in

both sexes; however, higher estrogen in girls explainsthe shorter period of longitudinal bone growth andultimate bone length in girls compared with boys(Fig. 15.4). The necessity of estrogen signaling forepiphyseal fusion has been demonstrated by the lackof epiphyseal fusion in men with aromatase deficiency

and ERα loss-of-function mutations.

Loss of Sex Steroids in the Adult Skeleton

At menopause, the ovaries cease to produce estro-gens, thus making peripheral production of estrogen,mainly through conversion of adrenal androgens inadipose tissue, the primary source of estrogen in post-menopausal women. In men, total testosterone gradu-ally declines by approximately 1% per year starting bythe third decade of life. In addition, the levels of sexhormone-binding proteins are markedly increased

with age in men, thus reducing the amount of bioavail-able testosterone.

The difference in bone loss between women andmen during aging is mainly attributable to the rapid

bone loss in women in the years immediately follow-ing menopause. In the first 510 years followingmenopause, women lose cancellous bone at a rate of approximately 46% per year and cortical bone at arate of approximately 12% per year. After this

period, women lose bone at a slower rate of 12% peryear in both compartments, which is similar to the rateof bone loss in men (Table 15.1). The rapid loss in theyears immediately after menopause leaves womenwith lower bone mass, but also with lower trabecularconnectivity and number, which makes their bonemore susceptible to fracture. Men also have approxi-mately three times greater periosteal expansion thanfemales during aging, which produces stronger bonegeometry. Decreased estrogen signaling also impairs

bone’s response to mechanical loading, which contri-

butes to bone loss.The mechanism for the slow rate of bone loss in

aging men is similar to that of the slow bone loss phasein women. Testosterone deficiency has some estrogen-independent effects on calcium absorption and bonecell functions; however, much of the effect of testoster-one deficiency on bone loss in men is related to theresulting estrogen deficiency and its consequences.

Changes in Bone Cells Induced by EstrogenDeficiency

The rapid rate of bone loss early after menopause inwomen results from a combination of the effects of loss of estrogens on different bone cells (Table 15.2).Estrogen deficiency leads to increased rate of bone turn-over and an imbalance in focal remodeling at the BMUlevel favoring bone resorption. There is overproductionof both osteoclasts and osteoblasts, accompanied by alonger life span of osteoclasts and a shorter life span of osteoblasts. The longitudinal extent of the BMU (which

TABLE 15.1 Bone Loss in Men and Women with Aging and Sex-Steroid Loss

Life Stage Compartment Rate of Loss Amount of Loss

510 years postmenopause Q Cancellous; 46%/years; Cancellous. cortical

Cortical 12%/years

Older age Q Cancellous; 12%/years; Cortical. cancellous

Cortical 12%/years

Older age R Cancellous; 12%/years; Cortical. cancellous

Cortical 12%/years

TABLE 15.2 Effects of Sex Steroid Deficiency on Bone Cells

Cell Type Number Birth Death

Osteoclasts Increased Increased Decreased

Osteoblasts Increased Increased Increased

Osteocytes Unknown Unknown Increased

Supply of osteoclasts exceeds demand.High rate of bone remodeling.




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is related to the lifetime of the BMU) is determined bythe supply of osteoclast and osteoblast precursors,whereas the depth of the BMU’s erosion lacunaedepends on the timing of apoptosis in mature osteo-clasts. In estrogen deficiency, the supply of osteoclastprecursors is enhanced, resulting in the origination of more BMUs per unit bone area (i.e. a higher activationfrequency), and there are more osteoclasts and osteo-

blasts contributing to extend the progression of eachBMU. Moreover, osteoclasts live longer, resulting indeeper resorption pits and delayed BMU reversal to theformation phase. Furthermore, osteoblast apoptosis isincreased and thus bone formation is disproportion-ately lower compared to resorption, contributing to anegative balance within each remodeling cycle andleading to bone loss. The prevalence of osteocyte apo-ptosis is also increased, adding to the bone fragility thatcharacterizes conditions of loss of sex steroids.

Effects of Estrogens and Androgens onOsteoclasts

Consistent with the increase in osteoclasts and boneresorption induced by sex steroid deficiency, estrogensand androgens decrease the number of osteoclastsin vivo and in vitro (Table 15.3). The cellular mecha-nism of reduction of osteoclasts involves inhibition of osteoclast generation combined with induction of oste-oclast apoptosis. Estrogens decrease the production of interleukin-1 (IL-1,) IL-6, and tumor necrosis factor(TNF-α) in cells that support osteoclast formation,resulting in inhibition of proliferation and the differen-tiation of osteoclast precursors toward mature osteo-clasts. The inhibitory effect of estrogens on cytokine

production is mediated by an interaction between theestrogen receptor and NF-κB and regulation of geneexpression mediated by this transcription factor(Table 15.3). Androgens exert similar effects as estro-gens on the production of pro-osteoclastogenic cyto-kines. In addition, estrogens induce apoptosis inmature osteoclasts by acting directly on these cells.Current evidence indicates that estrogens induce osteo-clast apoptosis by activating proapoptotic pathways,

including the mitogen-activated protein kinase(MAPK)-c-Jun N-terminal kinase (JNK) and TNF ligand superfamily member 6 (Fas ligand) pathways.

Effects of Estrogens and Androgens onOsteoblasts and Osteocytes

In contrast to their proapoptotic effect on osteo-clasts, estrogens and androgens inhibit apoptosis in

osteoblasts and osteocytes (Fig. 15.3; Table 15.3). Themechanism of this survival effect involves rapid activa-tion of survival kinases ERKs and PI3-K. This is fol-lowed by phosphorylation of the proapoptotic proteinBad, which leads to inactivation of the apoptotic prop-erties of the protein, and phosphorylation and activa-tion of the transcription factors ETS domain-containingprotein (Elk) and CCAAT/enhancer-binding protein

beta (C/EBP β), with subsequent changes in geneexpression. These kinase-mediated posttranslationaland transcriptional effects are required for estrogen-induced survival of osteoblasts and osteocytes.

GLUCOCORTICOIDS

Glucocorticoids are produced and released by theadrenal glands in response to stress. They regulatenumerous physiologic processes in a wide range of tissues. Among several effects, these hormones exertprofound immunosuppressive and anti-inflammatoryactions and induce apoptosis in many cell types,including T lymphocytes and monocytes. Because of these properties, exogenous glucocorticoids are exten-sively used for the treatment of immune and

inflammatory conditions, the management of organtransplantation, and as components of chemotherapyregimens for hematological cancers. However, long-term use of glucocorticoids is associated with severeadverse side effects in several organ systems. In partic-ular, prolonged use of exogenous glucocorticoids leadsto a dramatic loss of bone mineral and strength,similar to endogenous elevation of glucocorticoids inCushing disease.

TABLE 15.3 Effects of Estrogens on Bone Cells

Cell Type Effect of Estrogen Mechanism

Osteoclasts Induction of apoptosis (Fas ligand and ERK/JNKactivation)

Genotropic and nongenotropic

Stromal/osteoblastic cells andT lymphocytes

Inhibition of pro-osteoclastogenic cytokine production(IL-1, IL-6, and TNF α)

Genotropic, mediated by receptor-transcriptionfactor interaction

Osteoblasts and osteocytes Inhibition of apoptosis (ERKs and PI3-K) Nongenotropic

ERK, extracellular signal-regulated kinase; IL-1/6, interleukin-1/6; JNK, c-Jun N-terminal kinase; PI3-K, phosphoinositide 3-kinase.

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Epidemiology and Progression of Glucocorticoid-Induced Bone Disease

The prevalence of glucocorticoid-induced osteopo-rosis has changed markedly in recent years due to theincreased therapeutic use of these agents. Around1950, bone loss due to glucocorticoid excess was rareand more than 90% of the cases were due to endoge-nous hypercortisolism. Today, glucocorticoid-inducedosteoporosis is almost entirely an iatrogenic disorderand the most common cause of secondary osteoporo-sis. It occurs irrespective of the original disease beingtreated and all patients are susceptible, even if they donot present the usual risk factors for bone loss.

The loss of bone mineral upon glucocorticoidadministration is biphasic. BMD decreases rapidly at arate of 612% during the first year and more slowlythereafter, at a rate of approximately 3% per year. Atotal of 3050% of patients receiving long-term gluco-corticoid therapy present one bone fracture. The risk of fracture increases as much as 75% during the first 3months of treatment, before significant decreases inBMD are detected. Moreover, 25% of patients alsopresent with collapse of the femoral head associated

with osteonecrosis of the hip.

Glucocorticoids and Bone Cells

The bone fragility syndrome associated withglucocorticoid-induced osteoporosis is characterized

by a marked reduction in the number of osteoblasts

and the rate of bone formation (Fig. 15.5). Severalmechanisms account for this remarkable decrease in

bone formation, including reduced osteoblastogenesis,decreased activity of osteoblasts, and increased apo-ptosis in osteoblasts. In addition, the prevalence of osteocyte apoptosis is augmented with glucocorticoidtreatment. Mapping of apoptotic osteocytes demon-strates that they accumulate in areas juxtaposed to thesubchondral femoral bone that collapses in patientswith osteonecrosis, suggesting that osteocyte apoptosismight contribute to osteonecrosis and to the increase

in bone fragility (Fig. 15.5).The proapoptotic effect of glucocorticoids on osteoblasts and osteocytes results from direct actions of thesteroids on cells of the osteoblastic lineage, as the proa-poptotic effect of glucocorticoids is readily demonstra-

ble in cultured osteocytes and osteoblasts. Furthermore,transgenic mice overexpressing corticosteroid 11-beta-dehydrogenase isozyme 2, an enzyme that inactivatesglucocorticoids, in osteocytes and osteoblasts are pro-tected from glucocorticoid-induced apoptosis andchanges to bone mass and fragility.

The initial rapid bone loss induced by glucocorti-coid excess is also associated with increased osteoclasts

and elevated resorption (Fig. 15.5). This results froman inhibition of osteoclast apoptosis by glucocorticoidtreatment. In contrast, during the slower phase of boneloss seen with long-term treatment, osteoclasts are notincreased and may even decrease in number. This iscaused by decreased osteoclast generation resultingfrom reduction in the number of osteoblastic cells thatsupport osteoclast formation.

Glucocorticoids

in excess

Osteoclasts• early, transient increased resorption by

promoting osteoclast survival• later, decreased osteoclastogenesis

Osteocytes• increased osteocyte apoptosis

Decreased bone mass

Increased bone fragility

Osteoblasts and precursorsmarked decreased bone formation due to

• decreased osteoblastogenesis• increased osteoblast apoptosis• decreased synthetic capacity

FIGURE 15.5 Direct effects of glucocorticoidson bone cells. Effects of excess of glucocorticoidson osteoclasts, osteoblasts and their precursors, andosteocytes are summarized in the text boxes. Adapted

from Weinstein RS, 2011 N Engl J Med 365:6270.




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As a consequence of the individual effects of the hor-mones on the different bone cell types, glucocorticoid-induced osteoporosis is characterized by a normal orreduced number of osteoclasts and a markedly reducednumber of osteoblasts. These features are consistentwith a low bone remodeling condition, and contrastwith the high bone remodeling that ensues with loss of sex steroids or increased PTH production. This further

emphasizes that the effect of glucocorticoids on boneresults from direct action of the hormone on bone cells,rather than being a consequence of hypogonadismor secondary hyperparathyroidism, as previously

believed.

Glucocorticoid Receptors and DownstreamSignaling

The mechanism of glucocorticoid action involves binding to the glucocorticoid receptor, conformational

changes, and nuclear translocation of the ligand-boundreceptor, followed by cis or trans interactions withDNA and thereby induction or repression of genetranscription.

In addition, glucocorticoids exert actions indepen-dently of changes in gene transcription. Such actionsinclude modulation of the activity of intracellularkinases like ERKs, MAPK/JNK, and protein-tyrosinekinase 2-beta/focal adhesion kinase 2 (Pyk2; alsoknown as related adhesion focal tyrosine kinase, cellu-lar adhesion kinase, or calcium-dependent tyrosinekinase). Pyk2 is a member of the focal adhesion kinase(FAK) family of nonreceptor tyrosine kinases. Although

Pyk2 and FAK are highly homologous, they exhibitopposite effects on cell fate in fibroblasts, as well as inosteoblasts and osteocytes. Thus, whereas FAK activa-tion leads to cell spreading and survival, Pyk2 inducesreorganization of the cytoskeleton, cell detachment, andapoptosis. In particular, mechanical stimulation of osteoblasts and osteocytes promotes osteocyte survival

by activating FAK; and glucocorticoids promote osteo-cyte apoptosis by activating Pyk2 and MAPK/JNK,hence opposing FAK-induced survival. These changeslead to cell detachment-induced apoptosis (anoikis).

The proapoptotic action of glucocorticoids in cells of the osteoblastic lineage is exerted via a receptor-

mediated mechanism that induces rapid changes inkinase activity. However, apoptosis induced by gluco-corticoids is independent of new gene transcription.These mechanistic findings are consistent with in vivoevidence indicating that glucocorticoids can still sup-press bone formation in genetically modified mice inwhich glucocorticoid receptors are unable to dimerize,and thus cannot activate transcription.

THYROID HORMONE

Normal thyroid (or euthyroid) status is importantfor skeletal development, peak bone mass acquisitionduring growth, and bone maintenance in adulthood,as well as for normal bone mineralization. Conditionsof hypothyroidism or hyperthyroidism are both associ-ated with increased risk for fracture. Thyroid status iscontrolled by the hypothalamic-pituitary-thyroid axis.The hypothalamus secretes thyroliberin [thyrotropin-releasing hormone (TRH)], which stimulates theanterior pituitary gland to synthesize and release thy-rotropin [thyroid-stimulating hormone (TSH)], whichacts on the thyrotropin receptors [TSH receptors(TSHRs)] of thyroid follicular cells to stimulate theirgrowth and also synthesize and secrete the thyroidhormones thyroxine (T4) and 3,5,30-L-triiodothyronine(T3). T3 and T4 act on the pituitary gland and thehypothalamus to inhibit the synthesis and secretion of TSH and TRH, respectively, providing a negative feed-

back loop essential for maintaining thyroid status. T3and T4 are taken up into target cells by specific cellmembrane transporters. Within the target cells, T3 andT4 are metabolized by type II and III deiodinases.Type II deiodinase converts and activates T4 to T3 byremoving a 50 iodine from T4. Conversely, type IIIdeiodinase removes a 50 iodine to deactivate T3 by con-verting it to T2 and prevents conversion (and activa-tion) of T4 to T3 by instead converting it to theinactive reverse T3.

Active T3 freely translocates to the nucleus withintarget cells and binds to thyroid hormone receptors(TRs), of which there are three functional isoforms:

TRα1, TRβ1, and TRβ2. TRs are transcription factors of the nuclear receptor superfamily that heterodimerizewith the retinoid X receptor (RXR). The heterodimercontrols gene expression by interacting with thyroidhormone response elements in gene promoter regions.

Hypothyroidism during childhood results in delayedskeletal maturation and decreased stature, and exoge-nous T4 replacement therapy causes rapid catch-upgrowth, where normal adult height may be attained if treated early enough. An excess of thyroid hormone,named thyrotoxicosis, on the other hand, accelerates boneaging and reduces stature due to premature growthplate fusion. In adults, hypothyroidism increases the

length of the remodeling cycle by specifically prolongingthe formation and mineralization phases. This results inlow bone turnover and greater bone mass and minerali-zation. Hyperthyroidism decreases the length of theremodeling cycle and increases the frequency of initia-tion of remodeling, resulting in high bone turnover,

bone loss, reduced mineralization, and osteoporosis.Both hypo- and hyperthyroid conditions are associated

307THYROID HORMONE



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with increased fracture risk. Even in the healthy popula-tion, there is evidence to suggest that high-normal rangethyroid status is associated with reduced BMD andincreased risk of fracture, suggesting that thyroid statusaffects bone status in both physiologic and pathologicsituations.

Specific thyroid hormone transporters are expressedin osteoblasts, osteoclasts, and growth plate chondro-

cytes at different states of cell differentiation, indicat-ing that thyroid hormones can enter these cells. TRα1and TRβ1 are expressed in osteoblasts, osteoclasts,growth plate chondrocytes, and bone marrow stromalcells (BMSCs). It is unknown whether TRs areexpressed in osteocytes. Additionally, TSHR isexpressed in osteoblasts and osteoclasts, suggestingpotential direct effects of TSH in bone cells(Table 15.4).

In osteoblasts, T3 increases expression of alkalinephosphatase (ALP), fibroblast growth factor receptor 1(FGFR-1), insulin-like growth factor I (IGF-I), osteocal-cin, osteopontin, type I collagen, and matrix metallo-proteinases 9 and 13 (MMP-9 and MMP-13). In BMSCsand mature osteoblasts, T3 increases expression of IL-6,

IL-8, prostaglandin E2 (PGE2), and RANKL, which pro-mote osteoclastogenesis. It is unclear whether theeffects of T3 promote bone resorption only indirectlythrough osteoblastic mediation of osteoclastogenesis orwhether there are direct effects of T3 in osteoclasts. Theeffect of TSH on osteoblasts is unknown, as studieshave shown both inhibitory effects and stimulatoryeffects on osteoblastogenesis. Similarly, some studies

have shown an inhibitory effect of TSH on osteoclastsand bone resorption, but this has not been consistentlyobserved across all studies. T3 inhibits proliferationand promotes hypertrophic differentiation of growthplate chondrocytes. Therefore, in hypothyroidism,endochondral ossification and linear growth areimpaired, whereas in hyperthyroidism, endochondralossification is enhanced, resulting in short stature dueto premature closure of the growth plates (Table 15.4).

SOMATOTROPIN/GROWTH HORMONE

Somatotropin/GH and IGF-I are important regula-tors of bone during growth and throughout life. Many

TABLE 15.4 Effects of Triiodothyronine and Thyroid-Stimulating Hormone on Bone Cells

TR TSHR Effect of T3 Effect of TSH

Osteoblasts Yes Yes m Osteocalcin

m Osteopontinm Type 1 collagen

m ALPm IGF-1

m MMP-9/13m FGFR-1

m RANKLm IL-6/8m PGE2

Evidence for both stimulatory and inhibitory effects

Osteoclasts Yes Yes Indirect effects through osteoblastsDirect effects on osteoclast?

Inhibitory?

Growth plate chondrocytes Yes k Proliferationm Hypertrophic differentiation

Osteocytes ?

ALP, alkaline phosphatase; FGFR-1, fibroblast growth factor receptor-1; IGF-I, insulin-like growth factor I; IL-6/8, interleukin-6/8; MMP-9/13, matrixmetalloprotease 9/13; PGE2, prostaglandin E2; RANKL, RANK ligand/tumor necrosis factor ligand superfamily member 11; T3, 3,5,3 0-l-triiodothyronine; TR,thyroid hormone receptor; TSH, thyrotropin/thyroid-stimulating hormone; TSHR, thyrotropin/TSH receptor.




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of the effects of GH on bone are mediated throughlocal effects of IGF-I on bone, which are discussed inChapter 3. However, there is evidence for IGF-I-independent effects of GH on bone. For example, dou-

ble knockout mice deficient in GH receptor and IGF-Ihave a more severe bone phenotype than either singleknockout alone, indicating independent effects of thesehormones.

GH is a peptide hormone produced and secreted bythe somatotroph cells of the anterior pituitary gland.GH-releasing hormone (GHRH) stimulates and somato-statin inhibits the production and secretion of GH. Anegative feedback loop regulates GH, in which GH-stimulated hepatic IGF-I inhibits GH directly and alsoindirectly by stimulating release of somatostatin. GH isalso influenced by a number of other hormones, includ-ing ghrelin, sex steroid hormones, and thyroidhormone, which stimulate GH secretion, and glucocor-ticoids, which inhibit GH secretion. Additionally, GHstimulates PTH as well as 1α-hydroxylase responsiblefor 1,25(OH)2D3 production (Table 15.5). This interplayamong hormones makes it difficult to distinguish theeffects of GH on bone in various endocrine disorderswhere GH deficiency or excess occurs in conjunctionwith other hormonal abnormalities that may exert theirown effects on skeletal cells.

GH signals through the GH receptor (GHR), whichis a transmembrane receptor of the cytokine receptorsuperfamily. Upon GH binding, GHR dimerizes andsignals mainly through the JAK2/signal transducerand activator of transcription 1 (STAT) pathway butalso activates ERK1/2 and other MAPK pathways.

GH deficiency in humans is associated with low

BMD as well as low bone turnover, which is evident by histologic assessment of bone biopsies . GH defi-ciency that manifests during childhood is associatedwith short stature. Early age of onset and severity of GH deficiency determines the extent to which bone is

affected. Additionally, GH deficiency appears toaffect BMD in men more so than women. This sexualdimorphism may be attributable to differential effectsof concomitant hypogonadism on bone in men andwomen with GH deficiency. Limited data suggest anincreased risk of nonvertebral and vertebral fracturesin patients with GH deficiency, and that BMD isnot closely associated with fracture risk in this

population.On the other side of the GH spectrum, in acromeg-

aly, a disease usually caused by excessive GH secretion by a benign monoclonal pituitary adenoma, GH excesscauses bone overgrowth, with physical manifestationsincluding enlarged jaw bones, hands, and feet. Theseeffects, as well as higher BMD at cortical bone sites, areprobably due to effects on periosteal bone expansionPatients with acromegaly have increased bone turnoverwith a disproportionate increase in bone resorption,leading to bone loss, particularly at cancellous bonesites. Vertebral fractures are more common in acromeg-aly patients, partly due to lower vertebral BMD andpartly to vertebral deformities. Acromegaly can be trea-ted with surgical or pharmacological intervention toreduce GH levels.

As mentioned above, the GH-IGF-I axis is importantfor longitudinal growth and children with GH deficiencyhave short stature. Much of the effect of GH on chondro-cyte proliferation at the epiphyseal growth plate is medi-ated by hepatic and locally derived IGF-I. However, GHexerts direct effects on growth plate germinal layer pre-chondrocyte proliferation. Subsequently, GH-stimulatedlocal and circulating IGF-I increases the growth andproliferation of more mature growth plate chondrocytes.

Therefore, GH plays a primary role in directly stimulat-ing the proliferation of the prechondrocytes as an initiat-ing event for longitudinal growth, after which IGF-Isignaling predominates in continuation of chondrocyteclonal expansion (Fig. 15.6).

GH promotes osteoblastogenesis and bone forma-tion. GH stimulates proliferation of osteoblast lineagecells and also directs mesenchymal precursors towardthe osteoblastic and chondrocytic lineages over the adi-pocytic lineage. GH stimulates the expression of bonemorphogenic proteins, promoting osteoblast differenti-ation and bone formation (Fig. 15.6). The effects of GHon osteoclasts and bone resorption are less clear

because both stimulatory and inhibitory effects have been observed, which may in part be due to GHincreasing the production of OPG and IGF-I increasingthe production of RANKL by osteoblasts. In addition,IGF-I receptors are present in osteoclasts and directIGF-I signaling in osteoclasts may favor bone resorp-tion (Fig. 15.6).

In addition to effects of GH on bone and cartilagecells by direct and IGF-I-dependent mechanisms, GH

TABLE 15.5 Some Hormonal Interactions with GrowthHormone

Effect of GrowthHormone

Effect on GrowthHormone

m IGF-I k

m Sex steroids m Ghrelin m

Thyroid hormone m

Glucocorticoids k

m PTH

m 1,25-dihydroxyvitamin D

IGF-I, insulin-like growth factor I; PTH, parathyroid hormone.

309SOMATOTROPIN/GROWTH HORMONE



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may influence bone metabolism indirectly through itsactions on PTH, 1,25(OH)2D3, and phosphate handling.GH helps to maintain PTH secretion and circadianrhythm and increases the production of 1,25(OH)2D3

by increasing 1α-hydroxylase and inhibiting 24-hydroxylase. GH also increases phosphate retention byincreasing the renal maximal reabsorption thresholdfor phosphate. Together, these actions of GH favor

bone formation.

INSULIN

Insulin and IGFs are highly homologous, as aretheir receptors and their functions. The effects of IGFson bone are discussed in Chapter 3. In this chapter, themore direct effects of insulin on bone cells will bediscussed.

Insulin is a peptide hormone secreted by pancreatic beta cells in response to increased concentrations of glucose in blood. Insulin increases glucose uptake intotarget tissues and inhibits the release of stored energy.

Insulin (and IGFs) signals through the insulin receptor(IR), a cell surface tyrosine kinase receptor present intwo isoforms: α and β. IRs exist as either homodimersof the same IR isoform, or as heterodimers of IRα andIRβ or an IR with insulin-like growth factor-1 receptor(IGF-IR). Signaling transduction occurs by conforma-tional changes upon ligand binding, which result inautophosphorylation, followed by increased kinaseactivity of the receptor, and phosphorylation of a

number of substrate proteins that serve as effectormolecules.

Establishing the importance of insulin for boneindependent of IGFs is difficult due to their overlap-ping functions. However, type 1 diabetes mellitus(T1DM) patients, who are insulin deficient due to lossof pancreatic beta cell mass and function, have lower

bone mass and are at increased risk for early onsetosteoporosis and increased fracture risk. In addition,animal models of T1DM show that bone formation is

reduced, providing evidence for a relationship between insulin and bone, although these animals alsohave low circulating IGF-I.

IRs have been identified in osteoblasts, and treatingosteoblasts with insulin increases collagen synthesisand ALP activity. Global IR knockout mice are not via-

ble past the early postnatal period, but studies of cell-specific IR and IGF-IR deletion in osteoblasts have

been informative about the individual roles of insulinsignaling versus IGF-I signaling. These studies showthat diminished insulin signaling in osteoblasts resultsin reduced cancellous bone volume, with no defects inmineralization but reduced osteoblast number. On the

other hand, diminished IGF-I signaling in osteoblastsresults in reduced cancellous bone volume and under-mineralized bone, but with a normal number of osteo-

blasts. Cultured IR-deficient osteoblasts exhibitimpaired proliferation and differentiation, whereaswild-type osteoblasts treated with insulin haveincreased proliferation and differentiation (Fig. 15.7A).

More recently, insulin signaling in osteoblasts has been implicated in controlling whole body glucose

GH

MSC

Growth plate

prechondrocytes

↑ Proliferation

Longitudinal

growth

Adipocyte

lineageOsteoblast

lineage

↑BMPs

Chondrocyte

lineage

↑ Proliferation↑ Differentiation

↑OPG↑RANKLIGF-I

↑RANKL > ↑OPG?

Favors osteclastogenesisGH

Mature osteoblasts Mature osteoclasts

Bone formation Bone resorption

FIGURE 15.6 Effects of growth hormone on bonecells. Growth hormone (GH) directs mesenchymalstem cells toward chondrocytic and osteoblasticlineages and away from the adipocyte lineage. GHincreases osteoprotegerin (OPG) production, butthrough IGF-I also increases production of the RANKligand [tumor necrosis factor ligand superfamily mem-

ber 11/receptor activator of the NF-κB ligand(RANKL)], generally favoring osteoclastogenesis. GH

can also directly affect longitudinal growth by stimu-lating growth plate prechondrocyte proliferation. BMP, bone morphogenetic protein; IGF-I, insulin like growthfactor-I; MSC, mesenchymal stem cell; RANKL, tumornecrosis factor ligand superfamily member 11/receptoractivator of the NF-κB ligand.




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metabolism through an osteocalcin-dependent mecha-nism. Insulin signaling in osteoblasts increases theproduction of osteocalcin, which in turn acts on thepancreas to increase insulin production. Additionally,insulin signaling in osteoblasts decreases OPG andthus increases osteoclastic bone resorption. During

bone resorption, undercarboxylated osteocalcin, whichis considered the active hormonal form of osteocalcinregarding glucose metabolism, is liberated from the

bone matrix. This provocative animal experimentationdemonstrates a novel metabolic function of bone.However, the relative importance of insulin signalingin bone to overall glucose metabolism and the validity

of the hypothesis in humans remains to be determined(Fig. 15.7B).

1,25-DIHYDROXYVITAMIN D3

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3 or cholecal-ciferol] is a steroid hormone derived from vitamin Din the diet or from subcutaneous synthesis. Vitamin Dundergoes hydroxylation in the liver to produce 25(OH)D3, the serum indicator of vitamin D status, and asecond hydroxylation in the kidney to produce 1,25(OH)2D3, the hormonally active vitamin D metabolite.

1,25(OH)2D3 signals by binding to the vitamin D recep-tor (VDR), which is a member of the superfamily of nuclear receptors. VDR knockout mice develop hypo-calcemia, secondary hyperparathyroidism, and rickets,indicating a role for 1,25(OH)2D3 in bone mineraliza-tion. However, a diet high in calcium and phosphaterescues the abnormal mineral biochemistries and bonephenotype in the VDR knockout mouse, indicatingthat the main effects of 1,25(OH)2D3 on bone are to

provide sufficient calcium and phosphate for normalmineralization, particularly by mediating intestinal cal-cium and phosphate absorption. The role of 1,25(OH)2D3 on mineral homeostasis is discussed inChapter 13. Here, the direct effects of 1,25(OH)2D on

bone cells are discussed.VDR is present in cells of the osteoblastic lineage,

including osteoblast progenitor cells, osteoblast precur-sors, and mature osteoblasts. 1,25(OH)2D3 signaling inosteoblastic cells increases production of RANKL anddecreases the production of OPG, thus increasingRANKL-RANK-mediated osteoclastogenesis. This actionof 1,25(OH)2D3 is consistent with the actions of PTH and

1,25(OH)2D3 to increase serum calcium by liberating cal-cium from bone mineral.1,25(OH)2D3 signaling can also directly affect

bone formation. 1,25(OH)2D3 increases production of RUNX2, an essential transcription factor for osteoblastdifferentiation. Transgenic mice that overexpress VDRin osteoblastic cells have increased bone formation.Though the main role of 1,25(OH)2D3 in promoting

bone mineralization is through increasing intestinalcalcium and phosphate absorption (as evidenced bythe high-calcium/phosphate rescue diet in the VDRknockout mice), 1,25(OH)2D3 has also been shown tohave direct effects on osteoblasts to increase produc-

tion of osteocalcin and osteopontin, proteins involvedin bone mineralization (Fig. 15.8). Conversely, studieshave shown that high dose 1,25(OH)2D3 actuallyinhibits osteoblastic bone mineralization. Therefore,the direct effects of 1,25(OH)2D3 on bone are diverse,and can affect both bone resorption and formation pro-cesses. Its beneficial effects occur within a defined win-dow, and either high or low levels can be detrimentalto bone.

InsulinB A

Insulin

IR

IR ↑ Proliferation↑ Differentiation

↑ Collagen synthesis

↑ Osteocalcin↓ OPG

Osteoblasts

↑ Alkaline phosphatase

↑ Osteocalcin

Osteoblasts

↑ Bone formation and mineralization

↑ unOCPancreatic

beta cells

↑ Insulin↑ Insulinsensitivity

Insulin target tissues

FIGURE 15.7 Effects of insulin onosteoblasts and its proposed role in glu-

cose metabolism. Insulin increases prolif-eration and differentiation of osteoblasts(A) and increases collagen synthesis, boneformation, and mineralization. Insulinmay also act through an osteocalcin-mediated mechanism to regulate whole

body glucose homeostasis (B). IR, insulin

receptor; OPG, osteoprotegerin; unOC,undercarboxylated osteocalcin.

3111,25-DIHYDROXYVITAMIN D3



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LEPTIN

Leptin is a peptide hormone produced and secretedmainly by adipocytes. Leptin plays a role in energyhomeostasis, appetite, neuroendocrine function, immunefunction, reproduction capacity, and bone metabolism.Leptin exerts its effects by binding to leptin receptors,which are members of the class I cytokine receptorsuperfamily. Leptin receptors are expressed throughoutthe central nervous system and in peripheral tissues.Leptin signaling in the hypothalamus is important forenergy homeostasis. Congenital leptin deficiency resultsin obesity in both animal models and humans. Leptin

deficient (ob/ob) mice exhibit an obese phenotype that isrescued upon administration of leptin. Paradoxically,leptin excess is observed in obese individuals, appar-ently due to hypothalamic leptin resistance. Because themain source of leptin is adipose tissue, circulating leptinis highly correlated with body fat mass, particularly sub-cutaneous adiposity.

The effects of leptin on bone are complex, emergent,and dependent on dual-effects of central and periph-eral leptin signaling pathways (Fig. 15.9). A high bonemass phenotype has been characterized in leptin-deficient ob/ob mice, and intracerebroventricular infu-sion of leptin in both ob/ob and wild-type mice reduces

bone mass, suggesting that leptin decreases bone massthrough central mechanisms. However, the effect of leptin appears to vary by skeletal site. Thus, ob/ob micehave greater bone density and cancellous in the lum-

bar vertebrae, but they exhibit lower cortical bonedensity and volume in the femur. Due to the high con-tribution of cortical bone to total bone mass, ob/ob micehave reduced total body bone mass compared withwild-type mice. Therefore, central leptin signaling

appears to have dual effects on bone. Indeed, central

leptin effects mediated by β2-andrenergic receptorsdecrease osteoblast activity and bone formation andincrease remodeling of cancellous bone throughincreased RANKL, but central leptin effects mediated

by β1-andrenergic receptors or the GH/IGF-I axis stim-ulate bone formation, particularly at cortical sites(Fig. 15.9).

In contrast to intracerebroventricular infusion,peripheral administration of leptin increases bonemass in ob/ob mice. BMSCs, osteoblasts, and osteoclastsexpress leptin receptors. Leptin increases the expres-sion of osteogenic genes in BMSCs, leading to prefer-ential differentiation into the osteoblast lineage overadipocytes. In addition, leptin signaling in osteoblastsincreases the expression of OPG and decreases theexpression of RANKL, leading to decreased osteoclas-togenesis (Fig. 15.9).

Similar to adipocytes in peripheral body fat, adipo-cytes in the bone marrow also secrete leptin. Localeffects of leptin produced by bone marrow adipocytesadd another layer of complexity to the leptin-bonerelationship. In contrast to the peripheral effects of lep-tin on bone cells discussed above, higher concentra-tions of leptin stimulate bone marrow stromal cellapoptosis and bone resorption, and decrease bone

formation (Fig. 15.9). This suggests that a higher localconcentration of leptin from increased marrow adipos-ity may contribute to bone loss, a concept that is con-sistent with the positive association between marrowadiposity and osteopenia.

Body weight is positively associated with bonemass. This is commonly attributed to influences of mechanical stimulation from increased load-bearing.Leptin signaling on bone is also a potential

1,25-Dihydroxyvitamin D3

VDR VDR

↑ RANKL↓ OPG Osteoclastogenesis

↑ RUNX2

Osteoblast

lineage cellsMature osteoblasts

↑ Osteocalcin↑↑ Osteopontin

Bone mineralization

Mature osteoclasts

Bone resorption

↑ Serum Ca2+

FIGURE 15.8 Effects of 1,25-dihydroxyvitamin D3 on osteo-blast lineage cells and osteoclasts. In addition to its effects onintestinal calcium and phosphorus absorption (not shown), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] increases bone mineraliza-tion by driving differentiation of osteoblast lineage cells towardmature osteoblasts and by increasing osteoblast production of osteocalcin and osteopontin. Conversely, 1,25(OH)2D3 increases

bone resorption by increasing the RANK ligand/tumor necrosisfactor ligand superfamily member 11 (RANKL):osteoprotegerin

(OPG) ratio, thus promoting osteoclastogenesis. Ca

21

, calcium;VDR, vitamin D receptor.




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contributor to the higher bone mass observed withincreased body weight, as body weight is associatedwith bone mass even at “non-weight-bearing” sites.However, the high correlation between circulatingleptin and body fat in humans (accounting for more

than 80% of the variation in body fat) makes distin-guishing associations between leptin and bone fromassociations between body fat and bone challenging.Both positive and negative associations between cir-culating leptin and BMD have been reported, espe-cially when adjusted for body composition. Leptinreceptor polymorphisms have been associated with

bone mass in humans, but these associations may belargely mediated through leptin effects on energyhomeostasis and body size.

STUDY QUESTIONS

1. Describe how deficiencies of androgen and estrogenaffect bone at the cellular and structural levels.

Compare and contrast these effects on young and old

bone.

2. Describe how PTH can cause anabolism and

catabolism of bone.

3. Describe PTH signaling and its role in osteoblasts,

osteoclasts, and osteocytes.

4. Describe why hypothyroidism and hyperthyroidism

are both associated with fracture risk.

5. What are the roles of GH and IGF-I in the growing

skeleton?

6. How does obesity affect skeletal structure and

function?

Suggested Readings

PTH

Bellido, S., Divieti, in press. Effect of PTH on osteocytes. Bone.Dempster, D.W., et al., 1993. Anabolic actions of parathyroid hor-

mone on bone. Endocr. Rev. 14, 690709. Jilka, R.L., 2007. Molecular and cellular mechanisms of the anabolic

effect of intermittent PTH. Bone. 40, 14341446. Jilka, R.L., et al., 2008. Apoptosis of bone cells. In: Bilezikian, J.P.,

Raisz, L.G., Martin, T.J. (Eds.), Principles of Bone Biology, thirded. Academic Press.

Neer, R.M., et al., 2001. Effect of parathyroid hormone (134) onfractures and bone mineral density in postmenopausal womenwith osteoporosis. N. Engl. J. Med. 344, 14341441.

Obrien, C.A., 2010. Control of RANKL expression. Bone. 46,911919.

Sex Steroids

Kousteni, S., Bellido, T., et al., 2001. Nongenotropic, sex-nonspecificsignaling through the estrogen or androgen receptors: dissocia-tion from transcriptional activity. Cell. 104, 719730.

pp

↑ Bone resorption

↓ Cancellous bone↓ OB activity

↑ Bone remodeling

↑ OPG↓ RANKL

Body fat adipocytes

Local effects

of leptin in bone marrowLeptin

Leptin

Peripheral

signaling

Central

signaling

LEPRLEPR

Hypothalamus AdipocytesStromal cells

ApoptosisBone

marrow

Adipocyte

lineage

ADRβ2 ADRβ1 ↓ Bone formation

LEPR

osteoblasts

Osteoblasts

↑ Cortical boneformation

↓ Osteoclastogenesis ↑ Corticalbone formation

BMSC

Osteoblast

lineage

FIGURE 15.9 Concept model of central and peripheral effects of leptin on bone and local effects of leptin in bone marrow. Leptinsecreted by body fat adipocytes increases bone formation through peripheral signaling and has dual-effects on bone through central signaling.Leptin produced locally by adipocytes in the bone marrow increases stromal cell apoptosis, increases bone resorption, and decreases bone for-mation. BMSC, bone marrow stromal cell; LEPR, leptin receptor; OB, osteoblast; OPG, osteoprotegerin; RANKL, tumor necrosis factor ligandsuperfamily member 11/receptor activator of the NF-κB ligand.

313SUGGESTED READINGS


http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref1http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref1http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref1http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref2http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref2http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref2http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref3http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref3http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref3http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref4http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref4http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref4http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref4http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref4http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref5http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref5http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref5http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref6http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref6http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref6http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref6http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref6http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref6http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref6http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref6http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref5http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref5http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref5http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref4http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref4http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref4http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref4http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref4http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref3http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref3http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref3http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref2http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref2http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref2http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref1http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref1http://refhub.elsevier.com/B978-0-12-416015-6.00015-0/sbref1


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Manolagas, S.C., 2000. Birth and death of bone cells: basic regulatorymechanisms and implications for the pathogenesis and treatmentof osteoporosis. Endocrin. Rev. 21, 115137.

Nakamura, T., et al., 2007. Estrogen prevents bone loss via estrogenreceptor alpha and induction of Fas ligand in osteoclasts. Cell.130, 811823.

Riggs, B.L., Khosla, S., Melton third, L.J., 2002. Sex steroids and theconstruction and conservation of the adult skeleton. Endocr. Rev.23, 279302.

Glucocorticoids

Bellido, T., 2010. Antagonistic interplay between mechanical forcesand glucocorticoids in bone: a tale of kinases. J. Cell Biochem.111, 16.

Weinstein, R.S., 2011. Clinical practice. Glucocorticoid-induced bonedisease. N. Engl. J. Med. 365, 6270.

Weinstein, R.S., Manolagas, S.C., 2000. Apoptosis and Osteoporosis.Am. J. Med. 108, 153164.

Other Hormones

Fuzele, K., Clemens, T.L., 2012. Novel functions for insulin in bone.Bone. 50, 452456.

Giustina, A., Mazziotti, G., Canalis, E., 2008. Growth hormone, insulin-like growth factors, and the skeleton. Endocr. Rev. 29 (5), 535 559.

Gogakos, A.I., Bassett, J.H., Williams, G.R., 2010. Thyroid and bone.Arch. Biochem. Biophys. 503, 129136.

Hamrick, M.W., Ferrari, S.L., 2008. Leptin and the sympathetic con-nection of fat to bone. Osteoporos Int. 19, 905912.

Kawai, M., Devlin, M.J., Rosen, C.J., 2009. Fat targets for skeletalhealth. Nat. Rev. Rheumatol. 5 (7), 365372.

Mantzoros, C.S., Magkos, F., Brinkoetter, M., Sienkiewicz, E.,Dardeno, T.A., Kim, S.Y., et al., 2011. Leptin in human physiologyand pathophysiology. Am. J. Physiol. Endocrinol. Metab. 301 (4),E567E584.

Waung, J.A., Bassett, J.H., Williams, G.R., 2012. Thyroid hormonemetabolism in skeletal development and adult bone maintenance.Trends Endocrionol. Metab. 23, 155162.


4 HORMONAL AND METABOLIC EFFECTS ON BONE
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Hormonal Effects on Bone Cells

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