Kidney International, Vol. 63, Supplement 85 (2003), pp. S2–S5

Regulation of bone remodeling and emerging breakthroughdrugs for osteoporosis and osteolytic bone metastases

BRENDAN F. BOYCE, LIANPING XING, WILLIAM SHAKESPEARE, YIHAN WANG, DAVID DALGARNO,JOHN IULIUCCI, and TOMI SAWYER

University of Rochester Medical Center, Rochester, New York; and Ariad Pharmaceuticals, Cambridge, Massachusetts, USA

Regulation of bone remodeling and emerging breakthrough chondroclasts is still uncertain, but they may be in thedrugs for osteoporosis and osteolytic bone metastases. Major hematopoietic cell lineage. They are not required to beadvances have been made in the past 10 years in our under- osteoclasts because endochondral ossification is normalstanding of the molecular basis of bone cell formation and bone

or only slightly impaired in mammals that do not formremodeling. For example, the discovery of osteoprotegerin, theosteoclasts. The cartilage is replaced by cortical bonedecoy receptor and inhibitor of receptor activator of NF-�B

ligand (RANKL), and the RANKL/receptor activator of NF-�B formed by periosteal apposition and by trabecular bone,(RANK) signaling pathway that is essential for osteoclastogen- which is laid down within the medullary space at theesis, has helped clarify the mechanisms regulating osteoclast epiphyseal growth plate. Hematopoietic, stromal, andformation, activation, and survival. PTH, like most other osteo-

adipocytic bone marrow cells fill the remaining spaceclast stimulating factors, promotes RANKL production by os-within the bones. A network of endothelium-lined sinu-teoblast/stromal cells when they are exposed to it continuously,

but when given intermittently it stimulates bone formation and soids and feeding blood vessels nutritionally supportsreduces fracture risk in postmenopausal women. This anabolic these cells and the trabecular bone. Up to approximatelyeffect is associated with increased expression of insulin-like

30% of the volume of the space inside the bones of theand fibroblast growth factors and decreased osteoblast apopto-axial skeleton is composed of trabecular bone, whichsis. Src tyrosine kinase is essential for osteoclast activation and

also negatively regulates osteoblast activity. Thus, it is a well- contributes significantly to the supportive role of bonesvalidated therapeutic target for the prevention of postmeno- in the maintenance of normal posture. In contrast, tra-pausal and other forms of bone loss. Preliminary in vitro and becular bone is confined largely to the ends of most longin vivo studies of specifically designed, bone targeted, non-

bones, their diaphyseal cavities filled mostly with fattypeptide Src inhibitors have shown that these compounds inhibitmarrow in adult humans.bone resorption and stimulate new bone formation. The design

of drugs using structure/function approaches such as this shouldlead to the development of novel therapeutics that could be

BONE REMODELINGused to counteract the negative effects of chronic renal failureon the skeleton. Following bone modeling (growth), the integrity of

bones is maintained by the process of bone remodeling,in which worn-out sections of bones are removed byosteoclasts and replaced with new bone laid down byosteoblasts. Osteoclasts are multinucleated cells derivedBONE MODELINGfrom mononuclear precursors in the mononuclear-phago-Most bones are composed of an outer shell of protec-cyte lineage that fuse with one another by cytoplasmic,tive and supportive cortical (compact) bone, inside ofbut not nuclear, fusion [2]. Their formation and activitywhich is a network of trabeculae (plates) that compriseare regulated predominantly by osteoblasts and stromalthe cancellous, or spongy, part of bone. Most bones arecells. The stromal cells are derived from precursors informed during embryonic development by the processthe mesenchymal cell lineage that also gives rise to chon-of endochondral ossification, in which they are first mod-droblasts, adipocytes, fibroblasts, and muscle cells.eled in a mold or anlagen of cartilage [1]. Blood vessels

Osteoclasts remove packets (trenches) of bone (ap-invade the cartilage, which is resorbed by chondroclasts,proximately 60 �m deep) from the surfaces of bone tra-to form a medullary cavity. The precise origin of thesebeculae at remodeling sites, and osteoblasts fill in thesedefects by laying down and mineralizing new bone in a

Key words: osteoclast, osteoblast, RANKL, Src tyrosine kinase. process similar to that used to replace sections of dam-aged roadways. Thus, osteoclasts typically do not resorb 2003 by the International Society of Nephrology

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Boyce et al: Bone remodeling regulation and emerging therapies S-3

through the full thickness of trabecular elements duringnormal bone resorption and, consequently, leave a baseof bone matrix to which osteoblasts are attracted, andupon which they can lay down new bone matrix. Theresorption phase lasts up to approximately 30 to 40 days;the formation phase takes about 120 to 170 days [3].Recent studies have indicated that bone-lining cells inthe osteoblast/stromal cell lineage clean up the resorbedsurfaces and prepare them for new matrix deposition byremoving projecting collagen fibers and laying down athin layer of matrix to form the cement line that can beseen marking sites of resorption [4]. The collagen fibersare degraded by matrix metalloproteinases secreted bythese bone-lining cells, which do not appear to differenti- Fig. 1. RANKL signaling through TRAF 6 and the transcription fac-ate into the osteoblasts that subsequently fill in the re- tors, NF-�B and AP-1, in osteoclast precursors is essential for osteo-

clastogenesis. Signaling through Src, PI-3 kinase, and Akt mediatessorption cavity.cytoskeletal protein rearrangement and prevents apoptosis by phos-phorylating BAD and caspase 9, thus preventing the activation of theseenzymes, which mediate apoptosis. The osteoclastogenesis-specificREGULATION OF BONE RESORPTION genes that are regulated by NF-�B and AP-1 proteins, with the exceptionof NFAT2 [9], remain to be identified.Bone remodeling continues throughout life under the

influence a variety of factors, including mechanical strainand changes in the local and systemic levels of hormones,growth factors, and cytokines, all of which can stimulate RANK, on the surface of cells in the osteoclast lineagebone resorption. Our understanding of the mechanisms [2, 5]. It is secreted by other cells, including T lympho-whereby these factors can influence bone resorption dur- cytes, which thus can influence bone resorption at sitesing embryonic development and in pathologic states has of inflammation in and around bone and promote boneincreased considerably in the past decade, mainly as a loss [2, 5, 10]. RANK does not possess innate kinaseresult of studies of transgenic and knockout mice. These activity to mediate downstream signaling (Fig. 1) and,studies have helped identify the three most important like other members of the TNF superfamily, recruitsfactors elaborated by osteoblast/stromal cells that regu- TNF receptor-associated factors (TRAFs), which bindlate osteoclasts [2, 5]. These factors include macrophage- enzymes that have kinase activity [2, 5]. Among thesecolony stimulating factor (M-CSF), RANKL, and osteo- enzymes, Src tyrosine kinase plays an essential role inprotegrin (OPG). M-CSF commits CD11b-positive cells RANKL-mediated osteoclast activation because its ex-in the mononuclear/phagocyte lineage to the osteoclast pression is required for the cytoskeletal protein re-lineage by promoting expression of the receptor for arrangement that results in formation of the ruffled bor-RANKL, RANK on their surfaces. It also enhances os- der, the complex sea anemone-like change that takesteoclast activity by promoting their survival by pre- place in the surface membrane of the basal portion ofventing apoptosis. Although M-CSF expression is not the cell within the sealing zone and which attaches theessential for osteoclast formation, its absence is associ- cell tightly to the bone surface [11]. Within this sealingated with markedly diminished osteoclast formation and zone, hydrogen and chloride ions are secreted throughosteopetrosis, the hallmark of defective osteoclast for- proton and chloride pumps and combine to form hydro-mation or activation, which eventually is reversed in chloric acid, which dissolves the mineral in the boneop/op mice deficient in M-CSF by the complimentary matrix. Enzymes, including matrix metalloproteinasesaction of granulocyte macrophage-colony stimulating and cathepsin K, are secreted simultaneously to degradefactor (GM-CSF) [6]. the matrix. Src or other Src family members also appear

RANKL is a member of the tumor necrosis factor to mediate RANKL-mediated osteoclast survival in a(TNF) superfamily which enables the differentiation of signaling pathway that involves phosphorylation of PI3CD11b/RANK-positive cells along the osteoclast lineage kinase, Akt, BAD, and caspase 9, the last of which pre-and promotes their activation by enhancing polarization, vents activation of the apoptosis pathway [12, 13].ruffled border formation, and preventing apoptosis [7]. Full thickness resorption through trabecular elementsExpression of RANKL is essential for osteoclast forma- occurs in pathologic states, such as acute and chroniction, and its absence results in failure of osteoclast forma- inflammatory processes within the bone and at sites oftion and development of osteopetrosis [8]. Like M-CSF, osteolytic bone metastases. Resorption also accounts forRANKL is expressed on the surface of osteoblast/stro- the loss of trabecular elements that characterize post-

menopausal osteoporosis following sex steroid deficiencymal cells and interacts directly with its cognate receptor,

Boyce et al: Bone remodeling regulation and emerging therapiesS-4

and the associated increased risk of fracture observed These injections also increase the bone-sparing effectsof sex hormone replacement when given in combinationafter the menopause. Sex steroid deficiency and these

pathologic conditions are associated with increased pro- to postmenopausal women with established osteoporosis[21]. Intermittent PTH treatment does not appear toduction of proinflammatory cytokines, such as interleu-

kins 1 and 6, TNF�, and growth factors within the bone increase osteoblast proliferation on bone trabecular sur-faces, but appears to increase the bone formation ratemicroenvironment [14]. Most of these osteoclast-stimu-

lating factors stimulate bone resorption indirectly by pro- by inducing osteoblasts to increase the amount of matrixthey synthesize per day [22]. Recent studies have sug-moting the release of RANKL by osteoblast/stromal cells

within bone. RANKL release is also promoted by PTH gested that PTH may also increase osteoblast matrixproduction by preventing osteoblast apoptosis [23]. Thisand accounts for the osteoclast-stimulating activity seen

in hyperparathyroidism [2]. Interestingly, although in- action has also been attributed by Kousteni et al [24]to the osteoprotective effects of bisphosphonates andcreased PTH secretion also promotes increased RANKL

production by osteoblast/stromal cells, primary and sec- estrogen, the latter of which appear to be mediated bynongenotropic signaling.ondary hyperparathyroidism are not characterized by

full thickness resorption through trabecular elements. Posttransplantation bone loss is another major clinicalproblem that is associated with increased fracture riskThe basis for this difference in response to PTH and sex

steroid deficiency has not yet been explained fully, but [25] associated with increased bone resorption and inhi-bition of bone formation. This combination is also typicalit may reflect the fact that pro-inflammatory cytokines

activate NF-�B, a set of transcription factors involved of corticosteroid-related bone loss, which is a complicat-ing factor in posttransplant bone loss [26]. Thus, therein multiple cell functions [15]. NF-�B, in turn, can up-

regulate the expression of these cytokines, leading to the is an urgent need to develop new therapeutic agents thatnot only can prevent bone resorption, but also stimulateestablishment of an up-regulatory cycle in which sex

steroid deficiency leads to increased cytokine produc- new bone formation, particularly if their developmentcan be based on inhibition or stimulation of known func-tion, which in turn leads to increased NF-�B activity

and more cytokine release. Increased PTH secretion in tional signaling pathways in osteoclasts and osteoblasts.Src tyrosine kinase is a promising target for the devel-primary and secondary hyperparathyroidism is associ-

ated with increased blood concentrations of IL-6 [16], opment of drugs to prevent resorption and stimulate boneformation based on its known function in bone cells. Aspresumably through PTH-mediated osteoblast produc-

tion of IL-6 [17]. Although IL-6 can increase NF-�B stated earlier, Src expression is required for osteoclastactivation based on it role in cytoskeletal protein re-activity and, thus, potentially the subsequent production

of increased amounts of IL-1 and TNF�, there are no arrangement. More recent studies have indicated thatSrc also negatively regulates bone formation because Srcpublished studies indicating that this prevails in hyper-

parathyroidism. knockout mice have an enhanced osteoblast phenotypethat partly accounts for the increased bone mass observedIn addition to stimulating bone resorption, PTH also

has anabolic effects on bone. These are associated with in the mice [27]. However, because Src is expressed ubiq-uitously and has a regulatory role in multiple cell func-intermittent, rather than continuous, exposure to PTH,

which stimulates resorption and is followed by increased tions, including cell division, movement, and survival[28], tissue selectivity is likely to be an essential charac-bone formation, reflecting the coupling of bone forma-

tion to bone resorption. The precise mechanism by which teristic of an Src inhibitor, which, theoretically, couldinhibit bone resorption and stimulate bone formation.PTH has its anabolic effect remains poorly understood,

but it appears to be associated with up-regulation of Preliminary studies using Src tryosine kinase inhibitorsdeveloped at Ariad Pharmaceuticals (Cambridge, MA,insulin-like and fibroblast growth factors, potent stimula-

tors of bone formation [18, 19]. It is not known precisely USA), and targeted to bone by means of an incorporatedphosphonate-based chemical moiety show promising re-why continuous exposure to PTH is associated with in-

creased bone remodeling, while intermittent exposure sults in both in vitro and in vivo experiments [28].AP23451 is a lead compound Src kinase inhibitor devel-to PTH is not. It may be that intermittent increases in

PTH do not trigger an increased production of RANKL oped at Ariad that prevents PTH-induced hypercalce-mia and bone resorption, as well as ovariectomy- andby osteoblast/stromal cells and the subsequent NF-�B-

related up-regulation of osteoclastogenesis. metastatic breast cancer cell–induced bone loss [29, 30].AP23588 is a lead compound Src kinase inhibitor thatThe anabolic effects of PTH have been known for

many years and have been confirmed in recent studies not only prevents PTH-induced resorption, but also hasosteoblast stimulatory activity both in vitro and in vivoin rodents and humans [20]. These findings led to a num-

ber of recent clinical studies using recombinant PTH, [31]. The effects observed in rodents will require confir-mation in humans, but these preliminary findings suggestwhich have confirmed that daily injections of PTH in-

crease bone mineral density by approximately 14% [20]. that it should be possible to develop novel drugs based

Boyce et al: Bone remodeling regulation and emerging therapies S-5

11. Boyce BF, Yoneda T, Lowe C, et al: Requirement of pp60c-srcon these lead compounds, which have a dual action inexpression for osteoclasts to form ruffled borders and resorb bone

preventing bone loss and stimulating bone formation. in mice. J Clin Invest 90:1622–1627, 1992There are many other aspects of normal bone remod- 12. Wong B, Besser D, Kim N, et al: TRANCE, a TNF family member,

activates Akt/PKB through a signaling complex involving TRAF6eling that remain poorly understood. For example, theand c-Src. Mol Cell 4:1041–1049, 1999factors that direct osteoclasts or their precursors to sites 13. Xing L, Venegas AM, Chen A, et al: Genetic evidence for a role

destined for resorption remain to be identified. Osteo- for Src family kinases in TNF family receptor signaling and cellsurvival. Genes Dev 15:241–253, 2001cytes have been suggested as potential mediators of this

14. Manolagas SC, Kousteni S, Jilka RL: Sex steroids and bone.signaling because they have direct communication with Rec Prog Horm Res 57:385–409, 2002one another and with cells in the osteoblast lineage that 15. Baldwin A: The transcription factor NF-�B and human disease.

J Clin Invest 107:3–6, 2001line bone surfaces through dendritic processes extending16. Montalban C, Garcia-Unzueta MT, DeFrancisco AL, Amadowithin a canalicular network [32]. Studies involving mi- JA: Serum interleukin-6 in renal osteodystrophy: Relationship with

crodamage to bone using microstrain support the hy- serum PTH and bone remodeling markers. Hormone Metab Res31:4–7, 1999pothesis that damage to this communication network

17. deMiguel F, Martinez-Fernandez P, Guillen C, et al: Parathy-following fracture and breakage of the dendritic pro-roid hormone-related protein stimulates interleukin-6 expression

cesses is associated with osteocyte apoptosis and in- in human osteoblastic cells. J Am Soc Nephrol 10:796–803, 199918. Hurley MM, Marie P, Florkiewicz R: Fibroblast growth factorcreased bone resorption. Osteocytes have also been im-

(FGF) and FGF receptor families in bone, in Principles of Boneplicated in estrogen-mediated bone loss, which appearsBiology, edited by Bilezikian PJ, Raisz GL, Rodan AG, New

to be associated with increased osteocyte apoptosis. Un- York, Academic Press, Inc., 2002, pp 825–85119. Hock JM, Fitzpatrick A, Bilezikian JP: Action of parathyroidderstanding of the cellular and molecular regulation of

hormone, in Principles of Bone Biology, edited by Bilezikian JP,all aspects of bone cell function will be critical for theRaisz LG, Rodan GA, New York, Academic Press, Inc., 2002,

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effects of parathyroid hormone. Osteoporosis Int 13:267–277, 2002known functions of signaling molecules.21. Neer RM, Arnaud CD, Zanchetta JR, et al: Effect of parathyroid

hormone (1–34) on fractures and bone mineral density in post-Reprint requests to Brendan F. Boyce, M.D., University of Rochestermenopausal women with osteoporosis. N Engl J Med 344:1434–Medical Center, Department of Pathology and Laboratory Medicine,1441, 2001601 Elmwood Avenue, Box 626, Rochester, NY, 14642.

22. Dempster DW, Cosman F, Kurland ES, et al: Effects of dailyE-mail: brendan_boyce@URMC.Rochester.edutreatment with parathyroid hormone on bone microarchitectureand turnover in patients with osteoporosis: A paired biopsy study.

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