Skeletal remodeling in health and diseaseMone Zaidi

The use of genetically manipulated mouse models, gene and protein discovery and the cataloguing of genetic mutations have each allowed us to obtain new insights into skeletal morphogenesis and remodeling. These techniques have made it possible to identify molecules that are obligatory for specific cellular functions, and to exploit these molecules for therapeutic purposes. New insights into the pathophysiology of diseases have also enabled us to understand molecular defects in a way that was not possible a decade ago. This review summarizes our current understanding of the carefully orchestrated cross-talk between cells of the bone marrow and between bone cells and the brain through which bone is constantly remodeled during adult life. It also highlights molecular aberrations that cause bone cells to become dysfunctional, as well as therapeutic options and opportunities to counteract skeletal loss.

Terrestrial vertebrates require a skeleton for both locomotion and mineral homeostasis. The pelvic girdle of the ancient land vertebrate Panderichthys illustrates the shift in locomotory dominance from the pectoral to the pelvic region during the transition to life on land1. Likewise, vertebrates adopted a mineralized skeleton as a reservoir for calcium, with parathy-roid hormone (PTH) as the regulatory stimulus for the tight control of plasma [Ca2+]. Two distinct types of bone therefore emerged. Cortical bone, which is mainly present in long bone shafts and consists of concen-tric layers of mineralized hardened collagen, provides strength by being highly resistant to bending and torsion. Cancellous bone, in contrast, is found primarily in the axial skeleton and at the ends of long bones and is rigid but spongy, with a vast surface area created by an interconnecting trabecular meshwork.

Different cell lineages have emerged to serve distinct skeletal functions. Chondrocytes and osteoblasts, both of which are derived from bone mar-row mesenchymal stem cells, first construct and then model (shape) the skeleton, respectively, for maximal resilience. By contrast, the osteoclast, which is derived from the hematopoietic stem cell, maintains mineral homeostasis by resorbing the extensive surface of mainly cancellous bone. The careful balance between bone deposition and resorption is crucial for the proper development and maintenance of bone size, shape and integrity.

Molecular communication between osteoblasts and osteoclasts and between bone cells and other bone marrow cells is a fundamental mecha-nism that regulates bone formation and resorption with remarkable preci-sion. For example, the main switch for osteoclastic bone resorption is the receptor activator for NF-κB-ligand (RANK-L), a cytokine that is released by activated osteoblasts. Its action on the RANK receptor is regulated by osteoprotegerin, a decoy receptor, which is also derived from osteoblasts. Osteoclast-to-osteoblast cross-talk occurs mostly through growth factors, such as transforming growth factor-β (TGF-β), which are released from

the bone matrix during resorption. Gap junction proteins, such as connex-ins, contribute to direct cell-to-cell communication, and notch siganling regulates cross-talk at the level of the hematopoietic stem cell niche2–4. Lymphocytes also communicate with osteoclasts through mechanisms that are just beginning to be understood under the aegis of a new discipline — osteoimmunology.

Superimposed upon this cellular conversation is regulation by systemic hormones, such as PTH and estrogen, and cytokines, such as tumor necro-sis factor-α (TNF-α (TNF-α α). These molecules adjust the production of RANK-L and osteoprotegerin to regulate bone resorption, which in turn is coupled to changes in bone formation. Some cytokines also control bone forma-tion directly. Yet another layer of complexity arises from the conversation between bone cells and the brain, in which parallel hypothalamic lepti-nergic stimuli that are transmitted by sympathetic nerves and pituitary neurohormones regulate bone cell function.

Not only must bone formation and resorption be balanced quantita-tively, they must also be tightly coupled both in time and space. Although its underlying mechanism remains largely unknown, this tight coupling is a prerequisite for the repair of the microscopic skeletal damage that occurs as a result of constant terrestrial impact. When this coupling is lost, in pathologies ranging from systemic diseases, such as osteoporosis, to local bone destruction in cancer, net bone loss ensues. Each bone disease poses a public health hazard; hence the renewed interest in skeletal biology over the past decade.

This review highlights the tightly integrated, but separately regulated, molecular signals that govern skeletal remodeling. I discuss new infor-mation on the mechanisms of inherited and acquired disorders of both osteoblasts and osteoclasts. Finally, I review current and future therapies that either inhibit bone resorption or stimulate bone formation.

Osteoblasts and bone formationSelf-renewing mesenchymal stem cells give rise to osteoblasts, chondro-cytes, fibroblasts, myocytes or adipocytes. Each phenotypic transition requires an exacting, uninterrupted and asynchronous program of gene expression. However, despite this singularity of lineage commitment, there is significant plasticity in that mature cells can transdifferentiate into cells of other lineages.

The Mount Sinai Bone Program, Department of Medicine, Box 1055, Mount

Sinai School of Medicine, New York, New York 10029, USA.

e-mail: mone.zaidi@mssm.edu

Published online 6 July 2007; doi:10.1038/nm1593

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The initial step in osteoblastogenesis is the determination of a mes-enchymal stem cell to become an osteoprogenitor, which coincides with high levels of expression of hormone and cytokine receptors, including the PTH, prostaglandin, interleukin (IL)-11, insulin-like growth factor-1 (IGF-1) and TGF-β receptors. Wingless-ints (Wnts) and bone morpho-genetic proteins (BMPs) mainly drive these early events, and the helix-loop-helix proteins Twist and Id maintain proliferation. The cells then stop proliferating, express alkaline phosphatase, and begin to secrete type 1 collagen and non-collagenous matrix proteins. These active osteoblasts, which have a distinctive morphology, lie anchored mainly by cadherin-11 and N-cadherin to newly formed protein matrix or osteoid.

Although the triple-helical type 1 collagen molecule is the basic com-ponent of osteoid, many non-collagenous proteins with diverse functions support the protein matrix (Table 1). The relatively poorly understood process of matrix mineralization involves the initial extrusion of calcium-rich, nucleated vesicles, followed by the enzymatic cleavage of pyrophos-phate, probably by alkaline phosphatase, which enables the first, albeit imperfect, crystal nuclei to form. These grow by aggregation and the addi-tion of calcium ions, a process that is assisted by phosphoprotein kinases and phosphatases that regulate phosphorylation of the key nucleating phosphoproteins, bone sialoprotein and dentine matrix protein-1.

Improper collagen synthesis or inappropriate deposition impairs min-eralization, resulting in the class of genetic diseases known as osteogen-esis imperfecta. These diseases, which can be reproduced in genetically manipulated mice, cause skeletal pathologies that range, depending on the locus and severity of the mutation, from little or no deformity to absent cranial mineralization. The hallmarks of ostoegenesis imperfecta are osteopenia, recurrent fractures and deformity, but the phenotype can extend to other organs that contain type 1 collagen, including the teeth, sclera and middle ear bones. The deletion of non-collagenous proteins, in contrast, only sometimes causes an overt phenotype5.

Mature mineralizing osteoblasts become embedded into the secreted matrix and differentiate terminally to become osteocytes, a third basic cell type in bone. Osteocytes do not contain alkaline phosphatase, but produce osteopontins, among other bone matrix proteins. They communicate with each other through connexin-mediated gap junctions in fillipodial extensions within the canalicular network. Compartmentalized within the matrix, osteocytes sense and respond to changes in fluid flow arising from stress, strain or pressure6, but the entity that senses mechanical stimulation has not been identified. Apoptosis or injury-induced necrosis of osteocytes defines the spatial domains in which bone remodeling occurs6. A sum-mary of osteoblast function is presented in Figure 1a.

RANK-L

WNTLRP5/6

TCF/Lef

Stabilizedβ-catenin

DKKWif1sFRP

IGF-1

Boneformation

Neural Osteocyte

MEK/ERKPLC/IP3

collagen 1A geneYAP

HDACs 3,4,6NFAT2Osterix

NFAT2P

BMPIGF

TNF

RUNX2

RUNX2

Smads

BMPsTGF-β

PTH FGF

Ca++

Calcineurin

C/EBPδDCX3PPARγTWISTSTAT1Msx2Lef1 P300

CBPMORF

GRAR

AP-1Oct-1

Smad 1/5DLX-5C/EBP

Smurf1/2

Signals forresorption

Mec

ha

nose

nsing

Osteoblasts &bone-lining cells

Osteoclast

Collagen IAOsteopontinOsteonectin

MineralizationCalcium &phosphate

Osteoid

Dietaryintake

Vit. D

CREB

ATF4

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OPG

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+− Mechanical

stimulation

Sympathetic N

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IntermittentPTH

Ana

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Figure 1 Diverse functions and regulation of the osteoblast. (a) The key functions of osteoblasts are the formation of new bone and the regulation of osteoclastogenesis through RANK-L and osteoprotegerin (OPG). Osteoblasts also communicate with osteocytes to receive mechantransduction signals through gap junctional connexins. Intermittent PTH, IGF-1 and mechanotransduction are known extrinsic anabolic signals, whereas hypothalamic leptinergic signals transmitted through adrenergic nerves inhibit bone formation. (b) Three key intrinsic factors regulate osteoblast maturation and bone formation: the Runx2 platform for hormone and cytokine action (receptors not shown), with its assortment of cytosolic and nuclear co-activators (blue) and co-repressors (red); osterix, through its interaction with NFAT2; and the Wnt–β-catenin signals and their inhibitors (red). AR, androgen receptor; ATF-4, activating transcription factor-4; C/EBP, CCAAT/enhancer binding protein; CBP, CREB binding protein; DCX, double cortin; Dlx-5, distalless homeobox-5; FIAT, factor inhibiting activating transcription factor-4; GR, glucocorticoid receptor; HDAC, histone deacetylase; MORF, mortality factor; Oct-1; octamer binding protein-1; PLC, phospholipase C; PPAR, peroxisome proliferator-activated receptor; RSK, 90-kDa ribosomal S6 kinase; YAP, yes-associated protein.

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The intrinsic regulation of bone formation. An array of transcriptional regulators ensures the spatial and temporal precision with which discrete quanta of new bone are deposited. Mutations of these proteins gener-ally cause inheritable skeletal disorders, but because of their overlapping actions during endochondral ossification and adult bone formation, it is often impossible to separate embryonic from postnatal contributions to a skeletal phenotype. Loss-of-function phenotypes range from persisting cartilage and defective ossification, grouped as osseochondrodysplasias, to bone loss or osteoporosis. Gain-of-function mutations result in high bone mass, which is marked by autonomous, unrestricted and often dis-organized deposition of new bone. High bone mass is distinct from osteo-petrosis — a condition that results from osteoclastic defects and leads to poorly resorbed, brittle bone. Important transcriptional regulators are listed in Table 2, but for the purposes of this review, the focus is on the three main ones (Fig. 1b).

Runt-related transcription factor 2 (Runx2). The master transcription regulator for the osteoblast is Runx2, the expression of which is regulated negatively by the transcription factor Twist7. Runx2 is absolutely required for differentiation to proceed to the osteoblast and to no other lineage. As Runx2 also has a key function in endochondral bone formation, its deletion in mice or humans leads to a blend of cartilage and bony defects. That Runx2 exerts a dominant effect is demonstrated by the presence of overt cleidocranial dysplasia even during Runx2 haploinsufficiency. Subtle Runx2 polymorphisms likewise cause defects ranging from high bone mass8 to differences in femoral geometry9 to differences in femoral geometry9 to differences in femoral geometry .

Mechanistically, Runx2 binds to DNA with a partner, core binding fac-tor-β1 (CBF-β1; ref. 10), and serves as a platform for several cytokine and hormonal modifiers of osteoblast maturation. Because of its large assortment of binding partners and co-modulators (Fig. 1), Runx2 can be activated by several mechanisms. Phosphorylation of Smad by BMPs and TGF-β activates Runx2 as a crucial event in osteoblast maturation.

Mutations that disrupt the Smad-binding domain of the RUNX2 gene therefore produce more severe craniofacial deformity than other muta-tions, even when the Runt domain is preserved. By contrast, PTH and the fibroblast growth factors (FGFs) directly phosphorylate Runx2, and TNF leads to ubiquitination of Runx2 through the ligases Smurf-1 and Smurf-2 (ref. 11). However, despite the diverse molecular permutations that can activate it, Runx2 delimits multiple signals to achieve precise temporal homogeneity in inducing its osteogenic gene transcription program. This is because it forms multimeric complexes with DNA and its co-regulators in discrete nuclear matrix compartments. Molecules such as p300, and others with histone acetyl transferase activity, remodel chromatin to modify spatial domains sequentially and thus to specify defined gene expression profiles.

Osterix-NFAT interactions. Osterix (Osx) is another obligatory signal for osteoblast differentiation. Like Runx2, its expression is regulated by the anabolic signals BMP-2 and IGF-1, and like Runx−Runx−Runx /− mice, Osx-defi-cient mice do not form bone12. However, they have normal Runx2 levels, indicating that Osx is downstream of Runx2. Unlike Runx2, Osx interacts with the NFAT (nuclear factor for activated T cells) transcription factor family member NFAT2 (ref. 13) (Fig. 1b). Nonetheless, NFAT2 must first be activated through its dephosphorylation by the calcium/calmodulin-regulated phosphatase calcineurin. Activated NFAT2 then binds to Osx to stimulate osteoblastogenesis and bone formation13. Deletion of NFAT2 in the osteoblast therefore impairs bone formation, as does deficiency of the Aα isoform of calcineurinα isoform of calcineurinα 14.

Wnt–β-catenin signaling. Canonical Wnt–β-catenin signaling is another critical pathway for both skeletogenesis and skeletal remodeling. The first connections between Wnt signaling and bone came from the revelation that inactivating and activating mutations of the low-density lipoprotein receptor-related protein 5 (Lrp5) caused osteoporosis-pseudoglioma syndrome and a high bone mass phenotype, respectively. The high bone mass in the G171V (activating) mutation arises from a reduced affinity of the Lrp5 receptor for its inhibitor, the Dickkopf protein Dkk1, which allows unrestricted downstream signaling15. Another revelation is that, in contrast to the pure skeletal phenotype of RUNX2 mutations, LRP5 inac-LRP5 inac-LRP5tivation causes ophthalmic abnormalities and osteoporosis. Furthermore, unlike the dominance seen with the RUNX2 phenotype, Lrp5 haploinsuf-RUNX2 phenotype, Lrp5 haploinsuf-RUNX2ficiency causes only mild juvenile osteoporosis, without blindness16.

Lrp5, a single-transmembrane-domain receptor, interacts with the frizzled receptor complex to inhibit the phosphorylation of β-catenin by glycogen synthetase kinase-3β (GSK-3β). As phosphorylated β-catenin is more susceptible to ubiquitin-mediated degradation, by inhibiting GSK-3β activity, Lrp-5 allows the accumulation of β-catenin and its nuclear ingress. Activated β-catenin then interacts cooperatively with the Tcf/Lef

Table 1 Matrix proteins and their putative functionsProtein Regulatory role

Biglycan Collagen fibril formation

Decorin TGF-β activity

Osteonectin and tenasins Mineralization and matrix organization

Vitamin K-dependent Gla proteins(matrix Gla protein, osteocalcin, protein S)

Inhibitors of mineralization

RGD-containing glycoproteins (fibronec-tin, osteopontin, thrombospondin, bone sialoprotein, dentine matrix protein-1, vitronectin, fibrillin)

Osteoclast matrix interactions and/or mineralization

Table 2 Assortment of osteoblast transcriptional regulatorsRegulator Key features

Runx2 Platform through which multiple external signals are integrated; cooperates with Runx3 during skeletal development; activated upon downregulation of the leucine zipper protein Twist7; protein levels are regulated by Schnurri-3 by recruiting the E3 ligase WWP1 to Runx2 (ref. 121)

Osx Downstream of Runx212; interacts cooperatively with NFAT213

NFAT2 Cooperates with Osx13; unlike osteoclasts, not RANK-L regulated in osteoblasts

β-catenin Downstream of the Lrp5/Frz interaction with Wnt agonists; interacts with Tcf/Lef transcription factors

Smads Receptor Smads activated by binding of BMPs2/4/7 to BMPR1A/1B; heterodimerization with DNA-binding Smads; decoy receptors (noggin, DAN and chordin) prevent BMP overactivity21,122; define early osteoblastogenesis

AP-1 proteins c-fos and c-jun regulate early osteoblastogenesis; jun D and Fra-2 additionally control later osteoblast maturation

Homeodomain proteins Msx1 and Msx2 are early regulators of osteoblastogenesis; Dlx3 and Dlx5 also expressed later during osteoblastogenesis

ATF4 Substrate of Rsk2; regulates terminal differentiation to a mineralizing phenotype123; human mutation causes Coffin-Lowry syndrome

FIAT Leucine zipper protein that inhibits ATF4

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transcription factors to stimulate osteoblast differentiation. Mutations in Wnt members, ablation of the Wnt co-receptors Lrp5 or Lrp6, or use of Wnt inhibitors such as sclerostin, soluble frizzled-related proteins (sFRPs), Wnt inhibitory factors (Wif) and the Dickkoff family members, Dkk1 and Dkk2, reduce osteoblastogenesis and bone formation17–20. A monoclonal antibody against sclerostin has shown some promise in stimulating bone formation.

Osteoclasts and bone resorptionThe osteoclast is a large multinucleated cell, and is unique in its ability to resorb bone. Although its activity is normally integrated with the require-ments of skeletal morphogenesis and restructuring, excessive resorption

during adult life leads to bone loss, as occurs in osteoporosis, Paget’s bone disease, tumor osteolysis, various arthritides and periodontal disease. Below, I review the molecular pathways that regulate osteoclast develop-ment, function and survival, as well as genetic and acquired defects that cause human diseases.

Immune signals that regulate early osteoclast development. Multipotent hematopoietic stem cells in the bone marrow give rise to osteoclasts, macro-phages, lymphocytes and dendritic cells. Developmentally, therefore, the four lineages share regulatory mechanisms. There is also evidence of plas-ticity in that one cell type, such as a macrophage or B lymphocyte, can transdifferentiate into an osteoclast. Osteoclasts, however, are terminally

PU.1

c-fos,NF-κB

NFAT2JDP2,NF-κB

Determination

Proliferation,survival

Differentiation,survival

B-cellformation

b Differentiation

RANK

TRAF6

MKKsIKKs

IκB

NF-κB

JNK

NFAT2AP-1

ERK

DAP12

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Calcineurin

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Differentiationgenes

Ca++PSrc

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ERK1/2

c-fosAKT

GEFs

RacRho

c-FMS(M-CSF receptor)

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P

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c-cbl

Cyclin D

Actinreorganization

Grb2

a Proliferation

c Resorption

Ca2+

RyR

αvβ3

receptor

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SrcVav3

rac

CatK

Adaptors (cbl, etc.)

PLC-γ Grb2

MAPKs

MMPResorption pit

Matrix proteins

OCVN

OSPBSP

IL-6 transcription(negative feedback)

Src

Pyk2

Motility

Hydroxyapatite

cbl

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H+ Ca++Ca++

Adhesion

H+

H+

Dynamin

PI3K

Survival

c-fms

RANK

OSCAR

Resorption

RANK

αvβ3

Figure 2 Molecular mechanisms underlying osteoclastogenesis and bone resorption. (a) The early appearance of the M-CSF receptor c-fms places the determined precursor into a proliferation and survival mode that is mediated through the Akt and MAP kinase pathways, which converge to activate cyclin D. Also, the multi-site adaptor c-cbl acts as an ubiquitin ligase to allow cleavage of Bim-1, a pro-apoptotic cytokine113. (b) The action of RANK-L on its receptor, RANK, requires two co-stimulatory signals from the immunoreceptors TREM2 and OSCAR114. Once activated, RANK recruits the docking protein TRAF-6 to its cytoplasmic domain to activate NF-κB and MAP kinases. In parallel, the immunoreceptor tyrosine-based activation motif (ITAM)-harboringadapters DAP12 and FcRγ� recruit Syk kinases that activate phospholipase Cγ (PLC-γ (PLC-γ γ) to release Caγ) to release Caγ 2+ from intracellular stores22. The periodic release of Ca2+

activates the Ca2+/calmodulin activated phosphatase calcineurin, which then dephosphorylates the transcription factor NFAT2 (ref. 115). NFAT2 undergoes nuclear translocation to bind consensus sequences on DNA, and together with c-fos, amplifies key osteoclast genes, including NFAT2 itself116–118. (c) Mature osteoclasts first attach to bone through the interaction of the integrin αvβ3 with RGD motifs of matrix proteins. Outside-in signaling then leads to the formation of complexes that include the kinases c-src and Syk, and the guanine nucleotide-binding factor Vav-3 (ref. 119) to cause adhesion. Once adherent, the osteoclast polarizes: its bone-apposed ventral membrane is thrown into complex folds, the ruffled border, which harbors all secretory activity. Several molecular complexes form, rapidly dissociate and re-form to accomplish actin remodeling that underlies polarization and granule extrusion. For example, αvβ3 activates c-src and Pyk-2, which then recruit c-Cbl and Cbl-b, followed by PI3-kinase and the GTPase dynamin120. Likewise, complexes between gelsolin and integrin-associated proteins are formed, and various proteins including VASPs, ITAM-harboring proteins and c-src/Syk adaptors cooperate by unclear mechanisms. BSP, bone sialoprotein; catK, cathepsin K; ERK, extracellular signal–regulated kinase; FcRγ, Fc receptor γ; FSHR, FSH receptor; GEF, guanine nucleotide exchange factor; Grb2, growth factor receptor binding protein 2; JDP2, jun dimerization protein 2; JNK, Janus N-terminal kinase; MAPK, mitogen activated protein kinase; MEK, MAPK kinase; MKK, MAP kinase kinase; MMP metallproteinase; OC, osteocalcin; OSCAR, osteoclast-associated receptor; OSP, osteopontin; PI3K, phosphoinositide 3 kinase; Pyk2, proline-rich tyrosine kinase 2; RyR, ryanodine receptor; SOS, son of sevenless; TRAF, TNF receptor associated factor; TRP, transient receptor potential channels; VASP, vasodilator-stimulated phosphoprotein; VN, vitronectin.

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differentiated and hence retain their sophisticated lineage.The earliest step in osteoclastogenesis is determination of the stem cell

precursor that is induced by the B-cell transcription modulator PU.1. At around this point, the cells acquire the c-fms receptor and undergo prolif-eration in response to stimulation of that receptor by macrophage colony stimulating factor (M-CSF), using the molecular assortment shown in Figure 2a. The removal of src homology-2–containing inositol-5 phos-phatase (SHIP), a key negative regulator of M-CSF, results in abundant osteoclasts21.

Up to this point, B-lymphocytes can still be formed, but with the appear-ance of c-fos and the RANK receptor, the precursor becomes fully com-mitted to an osteoclast lineage. RANK-L is an absolute requirement for osteoclastogenesis. Thus, the genetic deletion of any molecule downstream of RANK (Fig. 2b) results in the abrogation of osteoclastogenesis. For example, like mice lacking RANK-L and those lacking RANK, both mice lacking spleen tyrosine kinase (Syk–/–Syk–/–Syk mice) and double mutants lacking –/– mice) and double mutants lacking –/–

both 12-kDa DNAX activation protein (DAP12) and the Fc receptor γsubunit lack osteoclasts and show osteopetrosis22. Furthermore, embry-onic precursors from NFAT2−/− mice fail to become osteoclasts ex vivo. Observations such as these establish each molecule as a new drug target. NFAT2, the most distal target, is not only indispensable but also sufficient for osteoclastogenesis, as its overexpression yields osteoclasts even in the absence of RANK-L23.

Genetic defects in RANK-L signaling. Inactivating mutations of RANK-L and RANK, which should, by analogy to their respective mouse pheno-types, cause dense ‘marble bones’ and affect the immune system, have not yet been identified in humans, although theoretically they should exist. Currently, human osteopetroses are restricted to mutations of the more distal molecules that regulate bone resorption. Nonetheless, con-stitutive activation of the RANK receptor causes at least three extremely rare autosomal-dominant systemic osteolyses, namely familial expansile osteolysis, the familial form of early Paget’s disease, and expansile skel-etal hyperphosphatasia24. As expected, autosomal-recessive inactivating mutations of the TNFSF11B gene that lead to osteoprotegerin deficiency cause juvenile Paget’s disease24. These childhood osteolytic diseases are

characterized by high-turnover bone loss, dental lysis, focal appendicular bone lesions and deafness.

Late resorption signals. An osteoclast creates a sealed microcompartment, akin to a phagolysosome. This is accomplished by a series of orchestrated molecular and physical changes (Fig. 2c). A vacuolar, nongastric H+-ATPase then pumps acid across the ruffled border, causing the ambient pH within the resorptive lacuna to fall to less than 4 (ref. 25). This allows acid-optimal enzymes, such as cathepsin K, to cleave the helical and telo-peptide regions of collagen and to release peptides that are transcytosed and extruded at the dorsolateral surface. These peptides are used clini-cally to determine the rates of bone resorption. A Cl–cally to determine the rates of bone resorption. A Cl–cally to determine the rates of bone resorption. A Cl current through the – current through the –

H+/Cl– antiporter ClC-7 balances proton extrusion and an HCO– antiporter ClC-7 balances proton extrusion and an HCO–3

–/Cl–

exchanger rectifies the resulting cellular alkalinization.Hydroxyapatite dissolution also elevates the ambient Ca2+ to ∼40 mM

(ref. 25). This activates a Ca2+ sensor on the osteoclast surface, causing the rapid release of Ca2+ from intracellular stores and capacitative Ca2+

influx26–28. A rising cytosolic Ca2+ activates endothelial nitric oxide syn-thase, which in turn, generates nitric oxide locally, allowing osteoclasts to detach and retract29. Re-establishment of resorption is accomplished by the Ca2+-mediated synthesis and local release of interleukin-6 from inhib-ited osteoclasts30. Interleukin-6 in turn delimits this inhibitory feedback, allowing osteoclasts to re-establish resorption30.

Although the physiology of Ca2+ sensing is well established, the sensor has not been identified. Homology cloning has failed to identify a mole-cule similar to the parathyroid Ca2+ sensing receptor (CaSR). Mice lacking the CaSR have normal osteoclasts, and drugs that modulate it fail to affect osteoclasts. A type 2 ryanodine receptor, located uniquely in the osteo-clast membrane, can serve as both a Ca2+ channel and a Ca2+ sensor27,31. Consistent with this, resorption is high in mice lacking the enzyme CD38, which converts NAD+ to cyclic ADPr, a physiological agonist of the ryano-dine receptor family32dine receptor family32dine receptor family . Also, as a sensor for NAD+ and by determining how weary an osteoclast becomes, CD38 activates Ca2+ sensing through cyclic ADPr to prevent further resorption33. Pro-osteoclastic cytokines, such as TNFα, upregulate CD38, probably to afford the redundancy required to protect the skeleton against excessive loss34.

Table 3 Systemic hormones regulating bone remodeling and bone massHormone Main action Key skeletal effects of global deletion

Estrogen Mainly anabolica Estrogen receptor-α/α/α β: osteoporosis from low formation64

GnRH: osteoporosis from low formation

Aromatase: severe osteoporosis from high resorption and low formation65

Testosterone Mainly anabolic Androgen receptor: osteoporosis from low formation124

1,25-(OH)2 vitamin D Pro-resorptive Vitamin D receptor: rickets rescued by dietary intervention

Thyroid hormone Pro-resorptive in excessb; anabolic during growth Thyroid hormone receptor-α: osteopetrosis from low resorption125

Thyroid hormone receptor-β: osteoporosis from high resorption124

Parathyroid hormone Pro-resorptive in excess; anabolic (intermittent) PTH: high density from low resorption104

PTH/PTHrPR: multiple cartilage defects

Growth hormone and IGF-1 Anabolic Growth hormone receptor: osteoporosis and runting

IGF-1R: osteoporosis and runting

IGF-1: osteoporosis and runting49

TSH Anti-resorptive TSHR: osteopenia in haploinsufficiency51

FSH Pro-resorptive FSHβ: normal/high density from low resorption52

FSH receptor: normal density from low resorption52 or low density from low formation63

Calcitonin Anti-resorptive Calcitonin: high bone mass56,57

Calcitonin receptor: bone formation defect58

aDirect osteoclastic effects of estrogen have been demonstrated in vitro only in limited studies. Importantly, estrogen also regulates skeletal homeostasis in men, with strong in vitro only in limited studies. Importantly, estrogen also regulates skeletal homeostasis in men, with strong in vitrocorrelations between bioavailable estrogen and bone remodeling. Gender-independent effects of estrogen have been shown to be nongenotropic.bThere is substantial in vitro evidence that thyroid hormones stimulate bone resorption; this has recently been confirmed by receptor deletion studiesin vitro evidence that thyroid hormones stimulate bone resorption; this has recently been confirmed by receptor deletion studiesin vitro 124. Thus, both low TSH and high thyroid hormones seem to contribute to bone loss in hyperthyroidism.

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Human osteopetroses. Unlike the high-bone-mass phenotypes dis-cussed above, which result from the over-deposition of bone, osteope-trosis arises from poor bone removal due to the absence of resorption components. It is seen in mice that are genetically deficient in H+-ATPase, C1C-7 or cathepsin K. In humans, the severest form is a malig-nant autosomal recessive variety arising from mutations in genes that encode the α3-subunit of the V-ATPase TCIRG1 or C1C-7. Resorption arrest leaves remnants of un-remodeled primary spongiosa, causing dense but brittle ‘marble’ bones, which can be severe enough to obliter-ate the marrow cavity and trap neural structures. A less severe form is seen in pycnodysostosis, a disease of dwarfism and fragile bones that results from a cathepsin K mutation. Interestingly, although the mani-festations of such mutations are predominantly skeletal and mostly recapitulated in null mice35, they can be accompanied by phenotypic characteristics that are unrelated to bone. For example, the absence of carbonic anhydrase II causes not only osteopetrosis, but also renal tubu-lar acidosis. Similarly, a mutation in IκB kinase-γ (IKKγ (IKKγ γ) that impairs signaling by the transcription factor NFκB produces osteopetrosis, as well as anhidrotic ectodermal dysplasia and immunodeficiency36. Some rare forms of osteopetrosis have still not been associated with a particular gene.

Cross-talk between bone and brainSuperimposed upon the conversation between the osteoclast, osteoblast and other cells in the bone marrow and the hierarchical control of this conversation by cytokines and hormones, there is now clear evidence for cross-talk between the brain and bone through two distinct routes37. The first consists of neuronal discharges originating from the hypothalamus (neural arm) and the second comprises hormonal signals arising from the pituitary (neurohumoral arm) (Fig. 3).

Neural arm. One of the most interesting revelations of the past decade has been the demonstration that the CNS directly regulates bone mass38,39. A key finding was that mice lacking either leptin (B6.V-Lepob/J or ob/ob mice) or its receptor (C57BL/6J-LeprdbLeprdbLepr or db/db mice) showed high bone mass due to increased bone formation. As expected from this, the stimulation of neurons expressing the leptin receptor by centrally administered leptin resulted in impaired bone formation and low bone mass, together establishing a role for a central leptinergic relay in skeletal regulation. Importantly, this anti-osteogenic action was independent of leptin’s known anorexogenic effect38. Consistent with this, mice lacking the leptinergic signals that regulate body mass, namely α-melanocyte stimulating hormone or cocaine- and amphetamine-regulated tran-script (CART), did not show a bone-related phenotype40. Moreover, lean leptin-deficient lipodystrophic mice had the same high bone mass as obese ob/ob mice. Finally, ob/ob mice showed high bone mass despite hypercortisolism and hypogonadism, substantiating the independence of central leptin action from glucocorticoids and sex steroids.

Another study firmly established that central leptinergic control of bone mass occurred through peripheral sympathetic discharges directed at the osteoblasts39. Thus, mice lacking adrenergic receptor β2 (Adrb2) or dopamine β-hydroxylase displayed high bone mass and lost the inhibitory effects of central leptin39,40. Blocking β-adrenergic signaling likewise increased bone mass39. Interestingly, however, Adrb2−/− mice showed not only enhanced bone formation, but also low bone resorp-tion, indicating that, in the absence of direct osteoclastic innervation, the neural effects on osteoclastic bone resorption were indirect, probably mediated by CART40. Furthermore, the egress of hematopoietic stem cell precursors of the osteoclast lineage from the hematopoietic stem cell niche was found to be controlled by adrenergic signaling through the osteoblasts41. Abrogation of neural discharges in demyelinated ceramide

galactosyltransferase-null mice prevented this egress41. The evidence is thus compelling that central leptinergic neurons and peripheral sym-pathetic nerves regulate not only osteoblastic bone formation, but also the genesis and function of bone-resorbing osteoclasts.

Most intriguing, however, is the finding that this adrenergic stimula-tion provides a circadian rhythmicity to bone mass by interacting with the clock genes Per and Per and Per Cry in osteoblasts, which cause anti-prolifera-Cry in osteoblasts, which cause anti-prolifera-Crytive effects by inhibiting G1 cyclin expression42. Although one would not intuitively expect a process as slow as bone remodeling to be under circadian control, these findings might explain the diurnal variation in bone resorption markers that has been noted in humans43.

It remains unclear whether the central regulation of bone mass is exerted exclusively through the leptinergic-sympathetic axis, or whether other neuronal systems, such as anti-cholinergic and peptidergic nerves, are also involved. Bone is endowed with Aδ- and C-type nerve fibers that carry several neurotransmitters, including neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP). CGRP has both anabolic and anti-resorptive actions44, whereas the role of NPY-containing nerve terminals remains inconclusive45,46. Regardless of whether leptin acts exclusively, it might be possible to block sympathetic discharges by selectively targeting bone with a β-blocker. It would be interesting to investigate whether patients receiving β-blockers have a higher bone mass and lower fracture risk than matched controls.

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Figure 3 Neural and neurohumoral regulation of bone mass. The neurohumoral arm regulates bone formation through the growth hormone/IGF-1 axis and resorption, using FSH, TSH and calcitonin. The neural arm regulates formation negatively through sympathetic discharges under hypothalamic leptinergic control, and resorption through CART (not shown), as well as by modulating the egress of osteoclast precursors from the hematopoietic stem cell niche. The dominant skeletal phenotype that results from the deletion of any component of this system indicates a lack of redundancy in these overlapping arms. From a systems standpoint, this dual regulation ensures both short- and long-term control. Whereas the neurohumoral arm mediates amplitude modulation through changes in plasma levels and so exerts continuous basal tonicity, rapid frequency-modulated neuronal firing, upon physical activity for example, probably overrides it. During disuse or mechanical off-loading, reduced neural firing might prevent the decrements in bone formation that would otherwise ensue.

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Neurohumoral arm. The neurohormones that are derived from the ante-rior pituitary include growth hormone, thyroid stimulating hormone (TSH) and follicle stimulating hormone (FSH). Calcitonin is a primitive neuropeptide. The key actions of traditional endocrine hormones, such as sex steroids, PTH, 1,25-dihydroxyvitamin D3 and thyroid hormone are summarized in Table 3, but are reviewed elsewhere47. The main skeletal consequences of deletion of each hormone or its receptor or synthetic enzyme are also shown in Table 3.

Growth hormone is anabolic, but exerts its effect by secreting IGF-1 from the liver. There is an epidemiological and genetic correlation between bone mass and serum IGF-1, rather than between bone mass and serum growth hormone48. Genetic IGF-1 deficiency causes profound growth retardation and osteopenia49. This skeletal loss is not reversed by growth hormone, confirming that IGF-1 is the active physiological stimulator of bone formation. The role of insulin receptors on osteoblasts is unclear.

Hitherto, the textbook view has been that TSH and FSH solely regulate the secretion of thyroxine and sex steroids from target endocrine organs50. However, recent studies show that TSH reduces the formation, function and survival of osteoclasts by acting on a G-protein-coupled receptor on osteoclasts51. That this skeletal effect is dominant and profound comes from the observation that mice with TSH receptor haploinsufficiency are osteopenic, despite having normal thyroid function and levels of circulating thyroid hormone51. Likewise, the idea that FSH stimulates osteoclast formation and function through FSH receptors on osteoclasts is consistent with the occurrence of high bone mass in FSHβ-haploin-sufficient mice, which have normal ovarian function52. Mechanistically, both TSH and FSH interact reciprocally with MAP kinases, NF-κB and Akt kinases downstream of RANK-L, although the precise molecular cascades remain unclear51,52. The two hormones also share a recipro-cal effect on the synthesis and secretion of TNFα: TSH inhibits TNFαproduction, whereas FSH stimulates it, actions that contribute to the anti- and pro-osteoclastic effects of the two hormones, respectively53,54. The pathophysiological and therapeutic implications of these data are discussed later in this review.

Calcitonin is a primitive neuropeptide whose origin dates back to the ascidian Styela clava55, but in mammals is produced from neurally derived C-cells of the thyroid gland. Even four decades after its discovery, and despite remarkable clarity regarding its receptor-mediated inhibition of osteoclasts, it is still not clear whether calcitonin is an anti-resorptive hor-mone. This is because its deletion yields a high bone mass rather than an osteopenic phenotype56,57. By contrast, mice deficient in amylin, another related peptide from the calcitonin gene superfamily, show osteopenia58, confirming earlier reports59. Furthermore, although calcitonin has been used as an anti-resorptive drug for more than two decades, we have not resolved the enigma that patients with medullary thyroid carcinoma, in whom calcitonin is grossly elevated, do not have high bone mass, and thy-roidectomized patients with virtually no calcitonin do not display severe osteoporosis. This discordance, despite its unclear mechanism, highlights partially understood evolutionary mechanisms that inhibit bone resorp-tion and maintain skeletal integrity with remarkable exactness. It is pos-sible that elevation of calcitonin during pregnancy, growth and lactation is essential for the skeletal conservation that has been noted in these periods of calcium stress. More recently, calcitonin has been shown to have struc-ture-modifying actions in models of arthritis60.

Bone loss mechanismsBone loss occurs when osteoblastic bone formation uncouples from osteo-clastic bone resorption, either temporally or spatially. An absolute increase in the rate of bone resorption, with a relative, but insufficient increase in the rate of bone formation, causes ‘high-turnover’ bone loss. This bone loss is generalized and is a hallmark of hypogonadism, thyrotoxicosis,

hyperparathyroidism and diseases of cytokine excess. However, when uncoupling is localized, focal osteolysis occurs, as in skeletal metastases, Paget’s bone disease, rheumatoid arthritis and periodontitis. In contrast to the hyper-resorption that characterizes high-turnover disease, bone formation can slow down per se and lag behind resorption, causing ‘low-per se and lag behind resorption, causing ‘low-per seturnover’ osteoporosis. This occurs as a result of aging, disuse, glucocor-ticoids and calcineurin inhibitors.

High-turnover bone loss. Fuller Albright first professed with remarkable clarity that the withdrawal of sex steroids causes bone loss61. The ensuing experimental and clinical evidence showing restoration of sex ster oids prevents bone loss led to the attribution of hypogonadal osteoporosis exclusively, almost as an aphorism, to sex hormone deficiency. Both of the defects that accompany hypogonadism — an early increase in bone resorption and the later, slower decrements in bone formation — have been attributed solely to the loss of estrogen action. Likewise, since Von Recklinghausen’s discovery of hyperthyroid bone loss, high thyroid hor-mones have been regarded as the sole culprits of this condition.

Studies from my laboratory suggest that although the reduced bone formation results from estrogen deprivation per se, elevated FSH causes the sharp early hyper-resorption that accompanies hypogonadism. Thus, mice lacking FSHβ or the FSH receptor, or hypogonadal (hpg) mice lack-ing gonadotrophin-releasing hormone (GnRH), which have no circu-lating FSH, or mice deficient in both estrogen receptors α and β with normal FSH levels, do not display the increases in bone resorption that would be expected to result from their severe hypogonadism52,62–64. In fact, osteoclastic resorption is attenuated (not enhanced) in some of these genotypes64. The less-than-expected osteopenia thus results from reduced bone formation arising from sex hormone deficiency62–64bone formation arising from sex hormone deficiency62–64bone formation arising from sex hormone deficiency . By contrast, when FSH is high, such as in aromatase-deficient mice or after ovariec-tomy, profound bone loss occurs due both to elevated bone resorption and reduced bone formation65. Hence, we believe that FSH is pro-resorptive and estrogen is primarily anabolic.

The pro-resorptive action of FSH has been substantiated in human studies. When matched for their estrogen levels, amenorrheic women with FSH levels >40 IU per liter suffer greater bone loss than amenor-rheic women with FSH levels <40 IU per liter (ref. 66). There are also strong correlations between serum FSH and bone mass during the early post-menopause67, in amenorrheic women with FSH levels >40 IU per liter (ref. 66) and after estrogen replacement. We suggest, therefore, that FSH probably causes the hyper-resorption that characterizes the early menopause and follows oophorectomy, aromatase inhibitor therapy or chemical menopause. Extrapolating from mice to humans, data showing that FSH haploinsufficiency in mice increases bone mass despite normal ovarian function52 lays the foundation for a future anti-FSH therapy to combat hypogonadal hyper-resorption. Such a therapy will be of particu-lar value during early menopause, as it could obviate estrogen regimens that increase the risk of breast cancer, but without further impairing ovar-ian function.

Patients with TSH receptor mutations that have been rendered clinically euthyroid show enhanced bone resorption and osteopenia68, as do mice that are haploinsufficient for the TSH receptor51. There is a tight corre-lation between serum TSH and fracture risk in hyperthyroid women69. Furthermore, recombinant TSH directly suppresses bone remodeling in post-menopausal women70 and in rats71. These findings indicate that TSH directly regulates bone mass, and that its deficiency contributes to the bone loss in hyperthyroidism.

As discussed above, both FSH and TSH reciprocally regulate the secre-tion of the proresorptive cytokine TNFα from macrophages and T lym-phocytes53,54. TNFα-transgenic mice, like TSH receptor-null mice, have severe high-turnover osteoporosis. Bone loss is abrogated when TSH

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receptor-null mice are rendered TNFα-deficient53. Moreover, like FSHβand FSH receptor-null mice52, mice lacking T lymphocytes or TNFαsignaling molecules do not show hypogonadal bone loss72. Thus, it is possible that TNFα is the crucial downstream mediator of the resorptive α is the crucial downstream mediator of the resorptive αeffects of high FSH and low TSH.

Hypogonadism triggers TNFα production from T lymphocytes, expands the T-cell repertoire in bone marrow, increases the thymic out-put of T cells and upregulates the B-cell-derived cytokine interleukin-7 (IL-7)72–74. It remains unclear whether these hypogonadal immune effects arise from estrogen deficiency per se, or whether they can be attributed to high FSH levels. It is also unclear whether these immune aberrations occur in hyperthyroid states with low TSH levels. If they do, it would provide an explanation for the pathophysiology of hypogonadal and thyrotoxic osteoporosis, whereby aberrant glycoprotein hormone secretion activates immune cells to cause bone loss.

With that said, immune cell activation can cause equally profound bone loss in the absence of aberrant glycoprotein hormone secretion. Severe localized and systemic bone loss due to enhanced osteoclast formation driven by T-cell-derived TNFα is best seen in rheumatoid arthritis, psoriasis and Crohn’s disease75. There is evidence that anti-TNF therapies can attenuate bone loss in rheumatoid arthritis and Crohn’s disease76, but it is unlikely that this approach will be used in hormone-deficient states, even if TNFα is proven to be the downstream signal. In contrast to TNFα, T-cell-derived interferon-γ inhibits osteoclast forma-γ inhibits osteoclast forma-γtion in systemic lupus erythrematosus, which is why the disease causes remarkably little bone loss77.

Low-turnover osteoporosis. The pathophysiology that underlies reduced bone formation during aging has remained an enigma, mainly because of the paucity of animal models that represent true aging, as opposed to accelerated senescence. Studies with the senescence-accelerated mouse, SAMP6, indicate that reduced osteoblastogenesis is the causal event78. By contrast, mice that are deficient in Klotho, an aging suppressor protein, primarily display reduced bone resorption that culminates in low bone formation79. Furthermore, although aged mice show altered levels of bone anabolic molecules and their inhibitors, such as IGF-BP2 and nog-gin, their contribution to age-related bone loss remains unclear80.

In contrast to aging, immunosuppressive therapy markedly suppresses bone formation81. This is relevant clinically when glucocorticoids are used together with the calcineurin inhibitors cyclosporine or FK506 for optimal immunosuppression after organ transplant. The ensuing acute, rapid and severe bone loss results in a fracture risk of up to 65%81. With calcineurin inhibitors alone, the early phase of high-turnover bone loss seems to be T-cell-dependent, as cyclosporine-induced osteopenia is abrogated in severe combined immune-deficient (SCID) mice82. The later, protracted low-turnover phase of bone loss arises from a direct inhibitory effect of both calcineurin inhibitors on bone formation, which is fully recapitulated in the calcineurin Aα knockout mouse14.

Lowered osteoblast differentiation is central to the action of glucocorti-coids, an effect that has been attributed, at least in part, to the stimulation of sFRP1, an inhibitor of the anabolic canonical Wnt pathway. Evidence that glucocorticoids stimulate osteoblast apoptosis by suppressing Wnt1, Wnt3a and Wnt5a is limited83. More recently, it has been shown that osteoclasts are necessary for glucocorticoid-induced osteoblast inhibi-tion84. The selective deletion of the glucocorticoid receptor in osteoclasts abrogates glucocorticoid-induced reductions in bone formation84. With this new discovery, it becomes essential, both on biological and clinical grounds, to identify the glucocorticoid-induced osteoclast-to-osteoblast signal. Nonetheless, this clinical imperative might be overshadowed by the future use of selective glucocorticoid receptor agonists (SEGRAs) that typically spare the skeleton85.

Current and future therapiesThe past decade has witnessed an exponential growth in human genet-ics, transgenesis, gene ablation, genomics and proteomics, all of which, when applied to skeletal biology, have unraveled complex mechanisms that maintain skeletal integrity with meticulous accuracy. Most promi-nent have been the discovery of the RANK-L/osteoprotegerin system; the comprehensive description of neural and neurohumoral mechanisms for bone mass regulation; a more thorough understanding of the transcrip-tional control of bone cell formation, function and fate; and the definition of a hitherto unrecognized discipline of osteoimmunology. Molecular mechanisms of skeletal morphogenesis and bone modeling have also been gleaned with equal pace, but the clinical consequences of inherited skeletal dysplasias continue to pose an important therapeutic challenge. The area of cancer and bone, a matter of clinical concern, is beginning to emerge into prominence, particularly with the first description of gene signatures that correlate with bone metastases86.

The clinical arena has expanded equally fiercely, with the realization that osteoporosis is an inevitable consequence of aging, and that the aging population will double over the next decade. The prevalence of osteopo-rosis is staggering, with ∼50 million affected and 1.5 million fractures per year worldwide87. The Study of Women Across Nations has shown that the most rapid bone loss ensues as early as the late peri-menopause67. This brings to the fore the need to expand diagnostic and treatment thresholds, which, in the future, will necessitate a much larger therapeutic armamen-tarium. The next decade is likely to witness a truer translational focus, aiming more towards drug discovery, particularly with the relentless iden-tification of new molecular targets.

Suppressing bone resorption below formation remains the goal of all currently approved anti-resorptive therapies for post-menopausal loss, including estrogens, the selective estrogen receptor modulator (SERM) raloxifene, bisphosphonates and calcitonin. In a low-turnover state, however, there is the inherent need to correct bone formation, for which an anabolic therapy might be preferred. However, to maintain skeletal integrity, bone formation must outweigh resorption, either absolutely, as with an anabolic treatment, or in relative terms, as with an anti-resorptive agent. Either action translates clinically into a stabilization of bone archi-tecture, increased bone mineral content, and reduced fracture risk, which is the ultimate clinical goal. Below, I discuss established and prospective anti-resorptive and anabolic agents.

Designer sex steroids. Estrogen and designer estrogens (or SERM) reduce osteoclast formation, not resorption per se. This mechanism explains the efficacy of raloxifene at reducing vertebral fractures in early post-meno-pausal women, as at this time osteoclastogenesis is escalating and trabecu-lar bone is being rapidly remodeled. Raloxifene fails to reduce fractures at nonvertebral, mainly cortical sites88, defining a narrow therapeutic window that is likely to carry over to the related agents lasofoxifene and bazedoxifene, both of which are currently in clinical trials. However, any reduction in breast cancer incidence will have relevance to their utiliza-tion in high-risk groups, such as those with a history or family history of breast cancer.

Designer estrogens bind to the same receptor in separate tissues to elicit different outputs by recruiting distinct co-regulators89. Tamoxifen, a prototype of tissue selectivity, becomes uterotrophic by recruiting the co-activators SRC-1, AIB1, CBP and Pax2 (ref. 90). By contrast, as an anti-estrogen to the breast, it recruits the co-repressors NCoR and SMRT. Conformational interactions between the activation function-2 domain and the most carboxyl terminal helix 12 of the estrogen recep-tor-α allow the receptor to switch between co-regulators, with a final output arising from thermodynamically optimal conformations defined by a cell’s complement of co-regulators91. A key challenge in designing

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bone-active SERMs will therefore be to identify co-repressors that are highly expressed in osteoclasts and to characterize nanoscale interactions between the receptor, ligand and co-repressor by computer modeling and crystallography. A further challenge will arise from the inherent cellular heterogeneity of breast cancer tissue, which makes cell-to-cell variations in co-regulator levels very likely. This could prevent a predictable and uniform anti-proliferative effect.

Bisphosphonates. Used initially as anti-corrosion agents in the chemi-cal industry, bisphosphonates are now the most widely used treatments for osteoporosis, tumor osteolysis, humoral hypercalcemia, multiple myeloma and Paget’s bone disease. The family has expanded to include several approved agents, namely pamidronate, alendronate, risedronate, ibandronate and zolendronate, which share a high affinity to exposed hydroxyapatite arising from their ‘bone hook’ (P-C-P core and R1 group). Their R2 group defines potency, with the highest potency being ascribed to molecules with a nitrogen-containing heterocyclic ring. Non-nitrogen-containing bisphosphonates form cytotoxic ATP metabolites, whereas the newer nitrogen-containing molecules inhibit certain mevalonate pathway enzymes. By binding competitively to the gerenyl pyrophosphate pocket of farnesyl pyrophosphate synthase, they block the prenylation of small GTP-binding proteins. Whether this represents the sole mechanism by which biphosphonates inhibit resorption remains debatable. It is also unclear how small doses of highly potent bisphosphonates produce long-lasting effects, so as to yield extended-interval dosing regimens. Possible explana-tions include recycling in bone, an action through γδT cells92 or effects on osteoclast formation. Even though their mechanisms are not fully under-stood, most bisphosphonates produce robust reductions in fracture risk, often within one year, by stabilizing trabecular connectivity93often within one year, by stabilizing trabecular connectivity93often within one year, by stabilizing trabecular connectivity .

Future anti-resorptive agents. Gene knock-out studies have shown that molecules whose ablation results in osteopetrosis, either without osteo-clasts (RANK-L) or with impaired osteoclasts (c-src and cathepsin K), constitute potential drug targets94. RANK-L signaling can be inhibited using osteoprotegerin, osteoprotegerin mimetics, RANK fragments or monoclonal antibodies against RANK-L or RANK. Although early studies provided proof-of-concept for the inhibition of resorption by osteopro-tegerin, it was soon realized that osteoprotegerin also binds the immune ligands TRAIL and TNF95 and is expressed in endothelial cells. When osteoprotegerin is deleted in mice, there is a peculiar arterial calcifica-tion96, and in humans, serum osteoprotegerin correlates with heart failure, coronary artery disease and cardiovascular mortality97,98coronary artery disease and cardiovascular mortality97,98coronary artery disease and cardiovascular mortality . Any lead osteo-protegerin mimetic will therefore require rigorous cardiovascular test-ing. By contrast, the anti-RANK-L monoclonal antibody AMG162, when injected every 6 months, inhibits resorption and increases bone mineral density with similar efficacy to alendronate99,100. Although it appears safe in clinical trials, there are concerns relating to potential long-term effects on lymphocyte and mammary gland maturation, defects that are pheno-copied in the RANK-L knockout mouse and that are unlikely to emerge in short-term trials.

Src is obligatory for bone resorption and permissive to osteoclast sur-vival and osteoblast differentiation. Despite controversy about whether the kinase activity is required for resorption101,102, both kinase and non-kinase inhibitors continue to be developed. Whether any src inhibitor is anabolic has yet to be determined; if so, such compounds will mimic strontium ranelate, which has both anti-resorptive and anabolic actions. Another target that is susceptible to dual intervention is the d2 isoform of the v-ATPase subunit V(0), the ablation of which causes both reduced osteoclast fusion and enhanced bone formation103. Finally, lead inhibitors of cathepsin K have shown promising preclinical results. However, despite enhanced mineral content and normal structure, the poorly resorbed bone

in cathepsin K-null mice is more fragile35 than that of controls.

Anabolic agents. The long-standing role of PTH as a pro-resorptive hor-mone was only recently confirmed by the demonstration of high bone mass in PTH-null mice104. However, when administered intermittently, PTH becomes anabolic by shifting osteoblasts away from RANK-L pro-duction toward osteogenesis (Fig. 1a). Although the targeted ablation of PTH-related protein (PTHrP) in osteoblasts might indicate that PTHrP, rather than PTH, is the anabolic stimulus105, the true physiology of PTH anabolism remains unclear. However, as intermittent PTH treatment reduces fracture risk, one could speculate that spiking endogenous PTH causes an anabolic advantage. PTH secretion in humans is tightly con-trolled by the CaSR and shows an interesting circadian rythm of 7 pulses per hour106. Short-acting allosteric CaSR modifiers or calcilytics should in principle be able to alter the frequency or amplitude of these pulses.

Among other anabolic targets, an anti-sclerostin monoclonal antibody is under development, which prevents the binding of the inhibitor sclerostin to Lrp5 and thereby allows canonical Wnt signaling to proceed. Although the Wnt pathway constitutes a potential therapeutic target, it poses sev-eral levels of complexity. First, different osteoblastic Wnts have different actions. For example, Wnt3a and Wnt5a prevent apoptosis83. However, Wnt10b makes a fate choice by not only activating the transcriptional regulators Runx2, Osx and Dlx5, but also suppressing the trancription factors PPAR-γ and C/EBP-γ and C/EBP-γ α to ensure that adipogenesis is prevented in α to ensure that adipogenesis is prevented in αfavor of osteoblast formation107. Second, while providing multiple targets, the ubiquity of Wnt signaling lends a level of redundancy that must be overcome. For example, Wnt3a is also upregulated in vascular pericytes that express the transcriptional regulator Msx2 (ref. 108). Increased osteo-genesis mediated by this mechanism is thought to underlie the vascular calcification seen in diabetes, dyslipidemias and end-stage renal disease. This cautions against a global Wnt approach for an osteoporosis therapeu-tic agent. Finally, in addition to effects on osteoblast maturation, β-catenin, cooperatively with the early B-cell factor EBF2, attenuates osteoprotegerin, thereby enhancing osteoclast formation109,110. β-catenin- or EBF2-null mice therefore have not only poor bone formation, but also increased resorption110. In fact, the conditional deletion of Tcf-1 or β-catenin in osteoblasts results in normal osteoblast numbers, but elevated resorption, owing to reduced osteoprotegerin production111. Thus, whereas Wnt tar-geting is likely to reverse the defects in both cell types, the relative effects on bone formation versus resorption will determine the outcome of any such therapeutic agent.

Finally, interest in statins continues unabated, as the drugs directly stim-ulate bone formation. However, no consistent relationship between fracture risk and statin use has been demonstrated112. This could be because statins undergo first-pass metabolism and have poor skeletal distribution.

The futureDespite their rapid emergence, the testing of new drugs may soon become challenging. The Food and Drug Administration currently requires any new agent to undergo a randomized double-blind placebo-controlled clin-ical trial with vertebral fracture reduction as primary end point. However, considering the changing standard of care, placebo-controlled studies for fracture reduction are no longer considered ethical. A comparator therapy must be used, which will inevitably require an unrealistic sample size or a surrogate nonfracture end-point. The current surrogates — bone mineral density and remodeling markers — do not provide information about bone structure or strength. However, the determination rather than extrapolation of true biomechanical strength must remain a paramount consideration, as bone with enhanced density and normal structure can still be brittle35. It is therefore possible that pharmaceutical companies will contemplate using placebo groups for pivotal clinical trials in nonindus-

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trialized countries. Their justification is likely to center on the standards of care prevalent in these countries, and the notion that at least some of those enrolled will benefit from the drug — an arguably ethical option. Despite this pessimism, the search for anti-osteoporosis agents will likely continue unabated.

“What drugs?” will remain a key question. Some argue that too many anti-resorptive agents already exist, and future efforts should concentrate toward new anabolic agents. Biologically, though, it is much simpler to inhibit bone resorption by shutting off one or more modes of osteoclast activity than it is to purposefully stimulate bone formation, while coupling it precisely to resorption. Furthermore, current experience indicates that both anabolics, such as PTH, and anti-resorptives, such as bisphospho-nates, reduce fracture risk by about 50–60%, a number that is not likely to change with future therapies, considering the array of nonskeletal fracture determinants. What will ultimately guide the creation of new drugs will be their ease of administration, an assurance of persistence and compli-ance and the absence of long-term side effects. In this respect, long-acting, highly targeted biological therapies against molecules that are not ubiqui-tously expressed are likely to emerge over the next decade.

ACKNOWLEDGMENTSI thank J. Iqbal (Medical Scientist Training Program student) for developing the figures, M.J. Sweeney for editorial assistance and L. Sun, B.S. Moonga, E. Abe and H.C. Blair for helpful critiques. I acknowledge the support of the US National Institutes of Health (grants AG14907, DK70526 and AG23176) and Department of Veteran Affairs (Merit Award and Geriatrics Research Education and Clinical Center).

COMPETING INTERESTS STATEMENTThe author declares competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/.

Published online at http://www.nature.com/naturemedicineReprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/

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