Birth and Death of Bone Cells: Basic Regulatory Mechanisms and Implications for the Pathogenesis and Treatment of Osteoporosis

STAVROS C. MANOLAGAS Division of Endocrinology & Metabolism, Center for Osteoporosis & Metabolic Bone Diseases, University of Arkansas for Medical Sciences, and the Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205, USA

ABSTRACT these cells. The purpose of this article is 3-fold: 1) to review the role The adult skeleton regenerates by temporary cellular structures and the molecular mechanism of action of regulatory molecules, such that comprise teams of juxtaposed osteoclasts and osteoblasts and as cytokines and hormones, in osteoclast and osteoblast birth and replace periodically old bone with new. A considerable body of evi- apoptosis; 2) to review the evidence for the contribution of changes in dence accumulated during the last decade has shown that the rate of bone cell birth or death to the pathogenesis of the most common forms genesis of these two highly specialized cell types, as well as the of osteoporosis; and 3) to highlight the implications of bone cell birth prevalence of their apoptosis, is essential for the maintenance of bone and death for a better understanding of the mechanism of action and homeostasis; and that common metabolic bone disorders such as os- efficacy of present and future pharmacotherapeutic agents for osteo- teoporosis result largely from a derangement in the birth or death of porosis. (Endocrine Reviews 21: 115–137, 2000)

I. Introduction B. Bisphosphonates and calcitonin II. Physiological Bone Regeneration C. Novel pharmacotherapeutic strategies A. Remodeling by the basic multicellular unit (BMU) XI. Summary and Conclusions III. Osteoblastogenesis and Osteoclastogenesis A. Growth factors and their antagonists B. Cytokines I. Introduction C. Systemic hormones And Athena lavished a marvelous splendor on the prince so that D. Adhesion molecules all the people gazed in wonder as he came forward. The elders IV. Reciprocal Relationship Between Osteoblastogenesis making way as he took his father’s seat. The first to speak was an and Adipogenesis old lord, Aegyptius, stooped with age, who knew the world by V. Serial and Parallel Models of Osteoblast and Osteoclast heart. Development VI. Function of the Mature Cells Homer, the Odyssey: translation by Robert Fagles A. Osteoblasts B. Osteocytes OSS OF height (stooping), Dowager’s hump, and ky- C. Lining cells L phosis are some of the most visible signs of old age in D. Osteoclasts humans. The primary reason for these involutional changes VII. Death of Bone Cells by Apoptosis is a progressive loss of bone mass that affects the axial (pri- VIII. Regulation of Bone Cell Proliferation and Activity marily trabecular) as well as the appendicular (primarily IX. Pathogenesis of Osteoporosis cortical) skeleton. Loss of bone mass, along with microar- A. Sex steroid deficiency chitectural deterioration of the skeleton, leads to enhanced B. Senescence bone fragility and increased fractures—the bone disease C. Glucocorticoid excess known as osteoporosis (1). Both men and women start losing X. Pharmacotherapeutic Implications of Osteoblast and bone in their 40s. However, women experience a rapid phase Osteocyte Apoptosis of loss during the first 5–10 yr after menopause, due to the A. Intermittent PTH administration loss of estrogen (2). In men this phase is obscure, since there is only a slow and progressive decline in sex steroid production; hence, the loss of bone in men is linear and slower Address reprint requests to: Stavros C. Manolagas, M.D., Ph.D., Center for Osteoporosis & Metabolic Bone Diseases, Division of (3). In addition to losing bone faster at the early postmeno- Endocrinology & Metabolism, University of Arkansas Medical School, pausal years, women also accumulate less skeletal mass than 4301 West Markam Street, Little Rock, Arkansas 72205 USA. E-mail: men during growth, particularly in puberty, resulting in [email protected] smaller bones with thinner cortices and smaller diameter. *Research from the author’s laboratory described in this review was supported by the NIH (P01-AG13918, R01-AR43003), the Department of Consequently, the incidence of bone fractures is 2- to 3-fold Veterans Affairs (merit grant and a research enhancement award pro- higher in women as compared with men (4). gram, REAP), and the 1999 Allied Signal Award for Research on Aging. In addition to sex steroid deficiency and the aging process

115 116 MANOLAGAS Vol. 21, No. 2 itself, loss of bone mass is accentuated when several other prevent accumulation of old bone. Remodeling, with positive conditions are present. The most common are chronic glucocor- balance, does occur in the growing skeleton as well. Its pur- ticoid excess (5), particularly its iatrogenic form, hyperthyroid- pose, quite different from those proposed for the adult skel- ism as well as inappropriately high T4 replacement, alcoholism, eton, is to expand the marrow cavity while increasing tra- prolonged immobilization, gastrointestinal disorders, hyper- becular thickness (12). calciuria, some types of malignancy, and cigarette smoking (6). Bone loss and eventually fractures are the hallmarks of A. Remodeling by the basic multicellular unit (BMU) osteoporosis, regardless of the underlying cause or causes. The bone loss associated with normal aging in women has Removal of bone (resorption) is the task of osteoclasts. been divided into two phases: one that is due to menopause Formation of new bone is the task of osteoblasts. Bone re- and one that is due to aging and affects men as well (7, 8). sorption and bone formation, however, are not separate, In elderly women these two phases eventually overlap, mak- independently regulated processes. In the uninjured adult ing it difficult to distinguish the effect of sex steroid defi- skeleton, all osteoclasts and osteoblasts belong to a unique ciency from the effect of the aging process itself. The effect temporary structure, known as a basic multicellular unit or of the aging process itself is also frequently obscured because BMU (13). Although during modeling one cannot distinguish of secondary hyperparathyroidism (9), resulting from im- anatomical units analogous to BMU per se, sculpting of the paired calcium absorption from the intestine with advancing growing skeleton requires spatial and temporal orchestra- age (Ͼ75 yr old). The bone loss that is due to glucocorticoid tion of the destination of osteoblasts and osteoclasts, albeit excess shares several features with the bone loss due to with different rules and coordinates to those operating in the senescence, but also has unique features of its own. None- BMU of the remodeling skeleton. The BMU, approximately theless, as is the case with the other types of bone loss, the 1–2 mm long and 0.2–0.4 mm wide, comprises a team of heterogeneity of the underlying conditions, some of which osteoclasts in the front, a team of osteoblasts in the rear, a (e.g., postmenopausal state, rheumatoid arthritis, etc.) inde- central vascular capillary, a nerve supply, and associated pendently contribute to skeletal deterioration, can distort the connective tissue (13). In healthy human adults, 3–4 million clinical and histological picture (10). Irrespective of the over- BMUs are initiated per year and about 1 million are operating lap, it is important to recognize that the pathogenetic mech- at any moment (Table 1). Each BMU begins at a particular anisms are quite distinct in the various forms of osteoporosis place and time (origination) and advances toward a target, and that sex hormone deficiency and aging have indepen- which is a region of bone in need of replacement, and for a dent effects. variable distance beyond its target (progression) and even- During the last few years, there have been significant ad- tually comes to rest (termination) (10). In cortical bone, the vances in our understanding of the pathogenetic mecha- BMU travels through the bone, excavating and replacing a nisms responsible for the bone loss associated with sex ste- tunnel. In cancellous bone, the BMU moves across the tra- roid deficiency, old age, and glucocorticoid excess. All these becular surface, excavating and replacing a trench. In both conditions do not cause loss of bone mass by turning on a situations, the cellular components of the BMUs maintain a completely new process. Instead, they cause a derangement well orchestrated spatial and temporal relationship with in the normal process of bone regeneration. Therefore, to each other. Osteoclasts adhere to bone and subsequently understand the pathogenesis of osteoporosis and rationalize remove it by acidification and proteolytic digestion. As the its treatment, one must first appreciate the basic principles of BMU advances, osteoclasts leave the resorption site and os- physiological bone regeneration. teoblasts move in to cover the excavated area and begin the process of new bone formation by secreting osteoid, which is eventually mineralized into new bone. The lifespan of the BMU is 6–9 months; much longer than II. Physiological Bone Regeneration the lifespan of its executive cells (Table 1). Therefore, con- The skeleton is a highly specialized and dynamic organ tinuous supply of new osteoclasts and osteoblasts from their that undergoes continuous regeneration. It consists of highly respective progenitors in the bone marrow is essential for the specialized cells, mineralized and unmineralized connective origination of BMUs and their progression on the bone sur- tissue matrix, and spaces that include the bone marrow cav- TABLE 1. Vital statistics of adult bone remodelinga ity, vascular canals, canaliculi, and lacunae. During development and growth, the skeleton is sculpted to achieve its ● Lifespan of BMU ϳ6–9 months shape and size by the removal of bone from one site and ● Speed ϳ25 ␮m/day ● Bone volume replaced by a single BMU ϳ0.025 mm3 deposition at a different one; this process is called modeling. ● Lifespan of osteoclasts ϳ2 weeks Once the skeleton has reached maturity, regeneration con- ● Lifespan of osteoblasts (active) ϳ3 months tinues in the form of a periodic replacement of old bone with ● Interval between successive remodeling events at the same new at the same location (11). This process is called remod- location ϳ2–5 years. ● ϳ b eling and is responsible for the complete regeneration of the Rate of turnover of whole skeleton 10% per year adult skeleton every 10 yr. The purpose of remodeling in the a From A. Michael Parfitt (13) b adult skeleton is not entirely clear, although in bones that are The 10% per year approximation for the entire skeleton is based on an average 4% turnover per year in cortical bone, which represents load bearing, remodeling most likely serves to repair fatigue roughly 75% of the entire skeleton; and an average 28% turnover per damage and to prevent excessive aging and its consequences. year in trabecular bone, which represents roughly 25% of the skeleton Hence, the most likely purpose of bone remodeling is to (0.75 ϫ 4 ϭ 3 and 0.25 ϫ 28 ϭ 7; 3 ϩ 7 ϭ 10). April, 2000 BIRTH AND DEATH OF BONE CELLS 117 face. Consequently, the balance between the supply of new the gene encoding an osteoblast-specific transcription factor, cells and their lifespan are key determinants of the number known as osteoblast specific factor 2 (Osf2) or core binding of either cell type in the BMU and the work performed by factor a1 (Cbfa1), hereafter referred to as Cbfa1 (24). In turn, each type of cells and are critical for the maintenance of bone Cbfa1 activates osteoblast-specific genes such as osteopontin, homeostasis. bone sialoprotein, type I collagen, and osteocalcin. The importance of Cbfa1 for osteoblasts has been highlighted by the evidence that knockout of the Cbfa1 gene in mice prevents III. Osteoblastogenesis and Osteoclastogenesis osteoblast development (25, 26). In addition to Cbfa1, BMP-4 Both osteoblasts and osteoclasts are derived from precur- induces a homeobox-containing gene, distal-less 5(Dlx5), sors originating in the bone marrow. The precursors of os- which also seems to act as a transcription factor, probably as teoblasts are multipotent mesenchymal stem cells, which a heterodimer with another homeobox-containing protein also give rise to bone marrow stromal cells, chondrocytes, (Msx2). Like Cbfa1, Dlx5 regulates the expression of osteo- muscle cells, and adipocytes (14–16), whereas the precursors blast-specific genes such as osteocalcin and alkaline phos- of osteoclasts are hematopoietic cells of the monocyte/mac- phatase, as well as mineralization (27–29). Other factors such ␤ ␤ rophage lineage (17, 18). Long before these cells could be as transforming growth factor (TGF ), platelet-derived cultured, the existence of multipotent mesenchymal stem growth factor (PDGF), insulin-like growth factors (IGFs), and cells was suspected (19), based on the evidence that fibro- members of the fibroblast growth factor (FGF) family can all blastic colonies formed in cultures of adherent bone marrow stimulate osteoblast differentiation (30, 31). However, ␤ cells can differentiate, under the appropriate stimuli, into whereas TGF , PDGF, FGF, and IGFs are able to influence the each of the above mentioned cells; these progenitors were replication and differentiation of committed osteoblast pro- named colony forming unit fibroblasts (CFU-F). When genitors toward the osteoblastic lineage, they cannot induce CFU-F are cultured in the presence of ␤-glycerophosphate osteoblast differentiation from uncommitted progenitor and ascorbic acid, the majority of the colonies form a min- cells. eralized bone nodule; these bone-forming colonies are In addition to growth factors, bone cells produce proteins known as CFU-osteoblast (CFU-OB) (16). Osteoblast progen- that modulate the activity of growth factors either by binding itors may originate not only from stromal mesenchymal pro- to them and thereby preventing interaction with their re- genitors of the marrow, but also pericytes — mesenchymal ceptors, by competing for the same receptors, or by promot- cells adherent to the endothelial layer of vessels (20). ing the activity of a particular factor. For example, osteoblasts Whereas osteoclast precursors reach bone from the circula- produce several IGF-binding proteins (IGFBPs). Of these, tion, osteoblast precursors most likely reach bone by migra- IGFBP-4 binds to IGF and blocks its action, whereas IGFBP-5 tion of progenitors from neighboring connective tissues. promotes the stimulatory effects of IGF on osteoblasts (30). The development and differentiation of osteoblasts and During the last few years, several proteins able to antagonize osteoclasts are controlled by growth factors and cytokines BMP action have also been discovered. Of them, noggin, produced in the bone marrow microenvironment as well as chordin, and cerberus were initially found in the Spemann adhesion molecules that mediate cell-cell and cell-matrix organizer of the Xenopus embryo and shown to be essential interactions. Several systemic hormones as well as mechan- for neuronal or head development (32–35). Noggin and chor- ical signals also exert potent effects on osteoclast and osteo- din inhibit the action of BMPs by binding directly and with blast development and differentiation. Although many de- high affinity with the latter proteins (36, 37). Such binding is tails remain to be established concerning the operation of this highly specific for BMP-2 and 4, as noggin binds BMP-7 with ␤ network, a few themes have emerged (21). First, several of the very low affinity and does not bind TGF or IGF-I. Addition growth factors and cytokines control each other’s production of human recombinant noggin to bone marrow cell cultures in a cascade fashion and, in some instances, form positive and from normal adult mice inhibits not only osteoblast, but also negative feedback loops. Second, there is extensive func- osteoclast, formation, and these effects can be reversed by tional redundancy among them. Third, some of the same exogenous BMP-2 (23). Consistent with this evidence, BMP-2 factors are capable of influencing the differentiation of both and -4 and BMP-2/4 receptor transcripts and proteins are osteoblasts and osteoclasts. Fourth, systemic hormones in- found in bone marrow cultures and in bone marrow-derived fluence the process of osteoblast and osteoclast formation via stromal/osteoblastic cell lines, as well as in murine adult their ability to control the production and/or action of local whole bone. Noggin expression has also been documented in mediators. all these cell preparations. These findings indicate that BMP-2 and -4 are expressed in the bone marrow in postnatal A. Growth factors and their antagonists life and serve to maintain the continuous supply of osteoblasts. The only factors capable of initiating osteoblastogenesis from uncommitted progenitors are bone morphogenetic pro- B. Cytokines teins (BMPs) (22). BMPs have been long implicated in skeletal development during embryonic life and fracture healing. Since the early stages of hematopoiesis and osteoclasto- More recently, it has become apparent that BMPs, and in genesis proceed along identical pathways, it is not surprising particular BMP-2 and –4, also initiate the commitment of that a large group of cytokines and colony-stimulating fac- mesenchymal precursors of the adult bone marrow to the tors that are involved in hematopoiesis also affect osteoclast osteoblastic lineage (23). BMPs stimulate the transcription of development (38). This group includes the interleukins IL-1, 118 MANOLAGAS Vol. 21, No. 2

IL-3, IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin mature osteoblast phenotype, characterized by increased al- M (OSM), ciliary neurotropic factor (CNTF), tumor necrosis kaline phosphatase and osteocalcin expression, and a con- factor (TNF), granulocyte macrophage-colony stimulating comitant decrease in proliferation (53, 54). Moreover, IL-6 factor (GM-CSF), M-CSF, and c-kit ligand. As opposed to the type cytokines stimulate the development of osteoblasts above mentioned cytokines that stimulate osteoclast devel- from noncommitted embryonic fibroblasts obtained from opment, IL-4, IL-10, IL-18, and interferon-␥ inhibit osteoclast 12-day-old murine fetuses (55). Consistent with the in vitro development. In the case of IL-18 the effect is mediated evidence, several in vivo studies have demonstrated in- through GM-CSF (39). creased bone formation in transgenic mice overexpressing IL-6 has attracted particular attention because of evidence OSM or LIF (56, 57). that it plays a pathogenetic role in several disease states TGF␤ is another example of a factor affecting both bone characterized by accelerated bone remodeling and excessive formation and bone resorption (58). Thus, in addition to its focal or systemic bone resorption (40). IL-6 is produced at ability to stimulate osteoblast differentiation, TGF␤ increases high levels by cells of the stromal/osteoblastic lineage in bone resorption by stimulating osteoclast formation. Injec- response to stimulation by a variety of other cytokines and tion of TGF␤ into the subcutaneous tissue that overlies the growth factors such as IL-1, TNF, TGF␤, PDGF, and IGF-II calvaria of adult mice causes increased bone resorption ac- (41–43). Binding of IL-6 or other members of the same cy- companied by the development of unusually large oste- tokine family (IL-11, LIF, OSM) to cytokine-specific cell sur- oclasts, as well as increased bone formation. The effects of face receptors (in the case of IL-6, the IL-6R␣) causes recruit- TGF␤ might be mediated by other cytokines involved in ment and homo- or heterodimerization of the signal osteoclastogenesis as TGF␤ can stimulate their production. transducing protein gp130, which is then tyrosine phosphor- Mice lacking the TGF␤1 gene due to targeted disruption ylated by members of the Janus family of tyrosine kinases exhibit excessive production of inflammatory cells, suggest- (JAKs) (44). This event results in tyrosine phosphorylation of ing that this growth factor normally operates to suppress several downstream signaling molecules, including mem- hematopoiesis (59). bers of the signal transducers and activators of transcription (STAT) family of transcription factors (45, 46). Phosphory- C. Systemic hormones lated STATs, in turn, undergo homo- and heterodimerization and translocate to the nucleus where they activate cytokine- The two principal hormones of the calcium homeostatic ␣ responsive gene transcription (47). The -subunit of the IL-6 system, namely PTH and l,25-dihydroxyvitamin D3 [1,25- receptor also exists in a soluble form (sIL-6R), but unlike most (OH)2D3], are potent stimulators of osteoclast formation (17, soluble cytokine receptors, it functions as an agonist by bind- 60). The ability of these hormones to stimulate osteoclast ing to IL-6 and then interacting with membrane-associated development and to regulate calcium absorption and excre- gp130 to stimulate JAK/STAT signaling (44). On the other tion from the intestine and kidney, respectively, are the key hand, the soluble form of gp130 blocks IL-6 action (48). elements of extracellular calcium homeostasis. Calcitonin, Alone or in concert with other agents, IL-6 stimulates the third of the classical bone-regulating hormones, inhibits osteoclastogenesis and promotes bone resorption. The cells osteoclast development and activity and promotes osteoclast that mediate the actions of the IL-6 type cytokines on oste- apoptosis. Although the antiresorptive properties of calcito- oclast formation appear to be the stromal/osteoblastic cells, nin have been exploited in the management of bone diseases as stimulation of IL-6R␣ expression on these cells allows with increased resorption, the role of this hormone in bone them to support osteoclast formation in response to IL-6 (49). physiology in humans, if any, remains questionable (61–63). These findings indicate that the osteoclastogenic property of PTH, PTH-related peptide, and 1,25-(OH)2D3 stimulate the IL-6 depends not only on its ability to act directly on hema- production of IL-6 and IL-11 by stromal/osteoblastic cells topoietic osteoclast progenitors, but also on the activation of (49, 64–66). Several other hormones, including estrogen, an- gp130 signaling in the stromal/osteoblastic cells that provide drogen, glucocorticoids, and T4, exert potent regulatory in- essential support for osteoclast formation. STAT3 activation fluences on the development of osteoclasts and osteoblasts in stromal/osteoblastic cells is essential for gp130-mediated by regulating the production and/or action of several cyto- osteoclast formation (50). Despite the effects of IL-6 on os- kines (21, 64, 67–69). teoclastogenesis in experimental in vitro systems, IL-6 is not required for osteoclastogenesis in vivo under normal phys- D. Adhesion molecules iological conditions. In fact, osteoclast formation is unaf- fected in sex steroid-replete mice treated with a neutralizing In addition to autocrine, paracrine, and endocrine signals, anti-IL-6 antibody, or in IL-6-deficient mice (51, 52). The most cell-cell and cell-matrix interactions are also required for the likely explanation for this is that the ␣-subunit of the IL-6 development of osteoclasts and osteoblasts (70–72). Such receptor in bone is a limiting factor, and that both a change interactions are mediated by proteins expressed on the sur- in the receptor and the cytokine are required for the IL-6- face of these cells and are responsible for contact between mediated increased osteoclastogenesis, seen in pathological osteoclast precursors with stromal/osteoblastic cells and fa- states. cilitation of the action of paracrine factors anchored to the IL-6 type cytokines are capable of influencing the differ- surface of cells that are required for bone cell development. entiation of osteoblasts as well. Thus, receptors for these Adhesion molecules are also involved in the migration of cytokines are expressed on a variety of stromal/osteoblastic osteoblast and osteoclast progenitors from the bone marrow cells, and ligand binding induces progression toward a more to sites of bone remodeling as well as the cellular polarization April, 2000 BIRTH AND DEATH OF BONE CELLS 119 of osteoclasts and the initiation and cessation of osteoclastic suppresses the muscle cell phenotype when transfected into bone resorption. More important, for the purpose of this G8 myoblastic cells (95). Taken together, these findings review, adhesion molecules play a role in the control of strongly suggest that PPAR␥2 plays a hierarchically domi- osteoblast and osteoclast development and apoptosis (73–77). nant role in the determination of the fate of mesenchymal The list of adhesion molecules that influence bone cell progenitors, due to its ability to inhibit the expression of development and function includes the integrins, particu- other lineage-specific transcription factors. Studies with a ␣ ␤ ␣ ␤ larly v 3 and 2 1, selectins, and cadherins, as well as a clonal cell line (2T3) suggest that BMP-2 induces osteoblast family of transmembrane proteins containing a disintegrin or adipocyte differentiation in mesenchymal precursors, de- and metalloprotease domain (ADAMS). Each of these pro- pending on whether the BMP receptor type IA or IB is ac- teins recognizes distinct ligands. For example, some integrins tivated. Therefore, BMP receptors may also play a critical role recognize a specific amino acid sequence (RGD) present in in both the specification and reciprocal differentiation of collagen, fibronectin, osteopontin, thrombospondin, bone osteoblast and adipocyte progenitors (96). sialoprotein, and vitronectin (78).

V. Serial and Parallel Models of Osteoblast and IV. Reciprocal Relationship Between Osteoclast Development Osteoblastogenesis and Adipogenesis Even though millions of small packets of bone are con- The cells that comprise the bone marrow stroma can serve stantly remodeled, bone mass is preserved thanks to a re- several diverse functions including support of hematopoiesis markably tight balance between the amount of bone resorbed and osteoclastogenesis, fat accumulation, and bone forma- and formed during each cycle of remodeling. In any estab- tion (79). This functional adaptation is apparently accom- lished BMU, bone resorption and formation are happening plished by the plasticity of some of the stem cell progeny as at the same time; new osteoblasts assemble only at sites exemplified by the ability of stromal cells to convert between where osteoclasts have recently completed resorption, a phe- the osteoblast and adipocyte phenotype. Thus, a stromal cell nomenon referred to as coupling, and formation begins to type known as the Westen-Bainton cell exhibits PTH recep- occur while resorption advances. The end result is a new tors and high alkaline phosphatase activity and gives rise to packet of bone, either a cylindrical osteon or Haversian sys- osteoblasts during fetal development and in hyperparathy- tem, or a plate-like hemiosteon, that has replaced the older roidism. On the other hand, when marrow hematopoietic bone that was removed (97). activity is reduced using chemotherapeutic agents, these As the BMU advances, cells are successively recruited at cells convert into adipocytes and can support myeloid cell each new cross-sectional location. Osteoblasts do not arrive production (80–84). Further, adipocytes isolated by limiting until the osteoclasts have moved on. However, during the dilution from cultures of rabbit bone marrow can form bone longitudinal progression of the BMU as a whole, new oste- in diffusion chamber implants (85). Conversely, addition of oclasts and new osteoblasts are needed simultaneously, al- certain fatty acids to cultures of osteoblastic cells causes them though not at the same location. Two models of osteoblast to differentiate into adipocyte-like cells (86). recruitment, a serial and a parallel (Fig. 1), can explain the It is likely that interconversion of stromal cells among distinction between the cross-sectional and longitudinal phenotypes, as well as commitment to a particular lineage events during BMU progression (38). According to the serial with suppression of alternative phenotypes, is governed by model, factors released from resorbed bone or the local in- specific transcription factors. Indeed, Cbfa1 is required for crease in mechanical strain resulting from bone resorption, commitment of mesenchymal progenitors to the osteoblast stimulate osteoblast precursor cell proliferation and differ- lineage. Mice that are deficient in this factor lack osteoblasts entiation (98–100). According to the parallel model, osteo- and mineralized bone matrix (26); and expression of Cbfa1 blast and osteoclast precursor proliferation and differentia- in fibroblastic cells induces transcription of osteoblast- tion occur concurrently in response to whatever signal specific genes (24). On the other hand, CCAAT/enhancer conveys the need for initiation of new BMUs, and whatever binding protein ␣ (C/EBP␣), C/EBP␤, and C/EBP␦, as well hormone prolongs their progression (10, 38). With either as peroxisome proliferator activated receptor ␥1 (PPAR␥1) model, new osteoblasts must be directed to the right location. and PPAR␥2 orchestrate adipocyte differentiation (87–90). Concurrent osteoblast and osteoclast production makes Introduction of C/EBP␣ in fibroblastic cells induces adipo- teleological sense as at least one of the means of maintaining cyte differentiation (91, 92), and transfection of fibroblastic a balance between bone formation and resorption under cells with PPAR␥2 and subsequent activation with an ap- normal conditions. In support of the existence of a parallel propriate ligand causes the development of adipocytes (93). pathway of osteoblast and osteoclast formation, it is well Using clonal cell lines isolated from the murine bone mar- established that osteoclasts cannot be formed in vitro unless row, it has been demonstrated that PPAR␥2 can convert appropriate stromal cells, analogous to the bone marrow stromal cells from a plastic osteoblastic phenotype that re- stromal cells that support hematopoiesis, are present to pro- versibly expresses adipocyte characteristics to terminally dif- vide essential support. The precise phenotype of the cells that ferentiated adipocytes. Moreover, PPAR␥2 suppresses the support osteoclast development remains unknown, but they expression of Cbfa1 and thereby osteoblast-specific genes are clearly related to both the osteoblast and the bone mar- (94). Similar to the inhibitory effect of PPAR␥2 on the os- row stromal/adipocytic lineages (101–104). Interestingly, teoblast phenotype, the combination of PPAR␥ and C/EBP␣ bone marrow-derived cells with both osteoblastic and adi- 120 MANOLAGAS Vol. 21, No. 2

FIG. 1. Serial and parallel models of osteoblast and osteoclast development. For explanation, please see text. PreOC, Preosteoclast; GF, growth factors released from the matrix of resorbed bone; preOB, osteoblast progenitors. The expressions of RANK ligand and RANK on preosteoblasts and preosteoclasts, respectively, are de- picted to indicate their critical contribution in osteoclastogenesis and thereby the dependency of osteoclastogenesis on preosteoblastic cells.

pogenic characteristics support the formation of osteoclasts, osteoclast progenitors, while RANK-ligand is expressed in but marrow-derived cells that exhibit a purely osteoblastic or committed preosteoblastic cells and T lymphocytes (108– adipocytic phenotype do not (94). More to the point, noggin, 110). RANK-ligand binds to RANK with high affinity. This a BMP antagonist, blocks not only osteoblastogenesis but interaction is essential and, together with M-CSF, sufficient also osteoclastogenesis in murine bone marrow cultures, in- for osteoclastogenesis. 1,25-(OH)2D3, PTH, PTHrP, gp130 ac- dicating that commitment of mesenchymal progenitors to the tivating cytokines (e.g., IL-6, IL-11), and IL-1 induce the ex- osteoblastic lineage is prerequisite for osteoclastogenesis pression of the RANK-ligand in stromal/osteoblastic cells (23). This evidence suggests that the early less differentiated (50, 107). Osteoprotegerin (OPG), the third of the three pro- progeny of common mesenchymal progenitors of the osteo- teins, unlike the other two, is a secreted disulfide-linked blastic and adipocytic lineage can support osteoclast devel- dimeric glycoprotein. A hydrophobic leader peptide and opment, but more differentiated cells that have committed to three and one-half TNF receptor-like cysteine-rich pseu- either the osteoblast or the adipocyte pathway lose this prop- dorepeats characterize the amino terminus of this protein. erty. It is possible, but as yet untested, that the cells that Unlike other members of the TNF receptor family, OPG does provide support for osteoclast development are a distinct not posses a transmembrane domain. OPG has very potent progeny of mesenchymal progenitors, which displays per- inhibitory effects on osteoclastogenesis and bone resorption manently a phenotype with mixed adipocytic/osteoblastic in vitro and in vivo (111). Consistent with an important role characteristics, but never progresses to a terminally differ- of OPG in the regulation of osteoclast formation, OPG trans- entiated osteoblast or adipocyte. For convenience and lack of genic mice develop osteopetrosis, whereas OPG knockout a better term, the cells that support osteoclast development mice exhibit severe osteoporosis (112). The antiosteoclasto- are frequently referred to as stromal/osteoblastic to indicate genic property of OPG is due to its ability to act as a decoy their similarities to both bone marrow stromal cells and os- by binding to RANK-ligand and blocking the RANK-ligand/ teoblasts. RANK interaction. In addition to skeletal metabolism, the In full agreement with the in vitro evidence for the de- RANK/RANK-ligand/OPG circuit may regulate several pendency of osteoclast development on support by cells re- other biological systems. Indeed, OPG is produced by many lated to osteoblasts, mice lacking osteoblasts due to Cbfa1 tissues other than bone, including skin, liver, stomach, in- deficiency also lack osteoclasts (26). In addition, marrow cells testine, lung, heart, kidney, and placenta as well as hema- from SAMP6 mice, a strain with defective osteoblastogenesis, topoietic and immune organs. Consistent with this, mice exhibit decreased osteoclastogenesis (105) and do not exhibit deficient in RANK-ligand completely lacked lymph nodes as the expected increase in osteoclastogenesis nor do they lose well as osteoclasts (113). Moreover, OPG is also a receptor for bone after loss of sex steroids (106). the cytotoxic ligand TRAIL (TNF-related apoptosis-inducing The molecular mechanism of the dependency of osteoclas- ligand) to which it binds with high affinity and inhibits togenesis on cells of the mesenchymal lineage has been elu- TRAIL-mediated apoptosis in lymphocytes (114) and also cidated during the last 2 yr with the discovery of three regulates antigen presentation and T cell activation (115). proteins involved in the TNF signaling pathway (reviewed Osteoblastic cells and T lymphocytes, the two cell types in Ref. 107). Two of these proteins are membrane-bound that express high levels of RANK-ligand, are also the two cell cytokine-like molecules: the receptor activator of nuclear fac- types that express high levels of the osteoblast-specific tran- tor-␬B (NF-␬B) (RANK) and the RANK-ligand. Other names scription factor Cbfa1 (24). More intriguingly, both the mu- used in the literature for RANK are osteoprotegerin ligand rine and human RANK-ligand genes contain two functional (OPG-L) and TRANCE. RANK is expressed in hematopoietic Cbfa1 sites, and mutation of these sites abrogates the tran- April, 2000 BIRTH AND DEATH OF BONE CELLS 121 scriptional activity of the RANK-ligand gene promoter (116). matrix, mature osteoblasts are essential for its mineraliza- Therefore, the cell-specific expression of RANK-ligand in tion, the process of deposition of hydroxyapatite (123, 124). cells of the stromal/osteoblastic lineage and concurrent dif- Osteoblasts are thought to regulate the local concentrations ferentiation of osteoblasts and osteoclasts might be dictated, of calcium and phosphate in such a way as to promote the at least in part, by interaction between an osteoblast-specific formation of hydroxyapatite. In view of the highly ordered, transcription factor and RANK-ligand. BMP 2 and -4 stim- well aligned, collagen fibrils complexed with the noncollag- ulate Cbfa1 expression. Based on these lines of evidence, it enous proteins formed by the osteoblast in lamellar bone, it has been postulated that the molecular underpinnings of the is likely that mineralization proceeds in association with, and control of the rate of bone regeneration and the concurrent perhaps governed by, the heteropolymeric matrix fibrils production of osteoclasts and osteoblasts could well be a themselves. Osteoblasts express relatively high amounts of BMP3Cbfa13RANK-ligand gene expression cascade in alkaline phosphatase, which is anchored to the external sur- cells of the bone marrow stromal/osteoblastic lineage (117, face of the plasma membrane. Alkaline phosphatase has been 118). According to this hypothesis, BMPs may provide the long thought to play a role in bone mineralization. Consistent tonic baseline control of both processes, and thereby the rate with this, deficiency of alkaline phosphatase due to genetic of bone remodeling, upon which other inputs (e.g., biome- defects leads to hypophosphatasia, a condition characterized chanical, hormonal, etc.) operate. by defective bone mineralization (125). However, the precise Studies with transgenic and knockout animal models as mechanism of mineralization and the exact role of alkaline well as models with spontaneous genetic mutations have phosphatase in this process remain unclear. Bone mineral- identified at least three transcription factors that are required ization lags behind matrix production and, in remodeling for osteoclast differentiation: PU-1, fos, and NF-␬b. A review sites in the adult bone, occurs at a distance of 8–10 ␮m from of the precise role of these factors is beyond the scope of this the osteoblast. Matrix synthesis determines the volume of article, but the reader is referred to a recent excellent review bone but not its density. Mineralization of the matrix in- of the topic (119). creases the density of bone by displacing water, but does not alter its volume.

VI. Function of the Mature Cells B. Osteocytes A. Osteoblasts Some osteoblasts are eventually buried within lacunae The fully differentiated osteoblasts produce and secrete of mineralized matrix. These cells are termed osteocytes proteins that constitute the bone matrix (120). The matrix is and are characterized by a striking stellate morphology, subsequently mineralized under the control of the same cells. reminiscent of the dendritic network of the nervous sys- A major product of the bone-forming osteoblast is type I tem (126, 127). Osteocytes are the most abundant cell type collagen. This polymeric protein is initially secreted in the in bone: there are 10 times as many osteocytes as osteo- form of a precursor, which contains peptide extensions at blasts. Osteocytes are regularly spaced throughout the both the amino-terminal and carboxyl ends of the molecule. mineralized matrix and communicate with each other and The propeptides are proteolytically removed. Further extra- with cells on the bone surface via multiple extensions of cellular processing results in mature three-chained type I their plasma membrane that run along the canaliculi; os- collagen molecules, which then assemble themselves into a teoblasts, in turn, communicate with cells of the bone collagen fibril. Individual collagen molecules become inter- marrow stroma which extend cellular projections onto connected by the formation of pyridinoline cross-links, endothelial cells inside the sinusoids. Thus, a syncytium which are unique to bone. Bone-forming osteoblasts synthe- extends from the entombed osteocytes all the way to the size a number of other proteins that are incorporated into the vessel wall (128) (Fig. 2). As a consequence, the strategic bone matrix, including osteocalcin and osteonectin, which location of osteocytes makes them excellent candidates for constitute 40% to 50% of the noncollagenous proteins of mechanosensory cells able to detect the need for bone bone. Mice deficient in osteocalcin develop a phenotype augmentation or reduction during functional adaptation marked by higher bone mass and improved bone quality, of the skeleton, and the need for repair of microdamage, suggesting that osteocalcin functions normally to limit bone and in both cases to transmit signals leading to the ap- formation without compromising mineralization (121). Con- propriate response; albeit this remains hypothetical (129). versely, mice deficient in osteonectin exhibit decreased os- Osteocytes evidently sense changes in interstitial fluid teoclast and osteoblast numbers and bone remodeling and flow through canaliculi (produced by mechanical forces) profound osteopenia, suggesting that, under normal condi- and detect changes in the levels of hormones, such as tions, this protein may play a role in the birth or survival of estrogen and glucocorticoids, that influence their survival these cells (122). Other osteoblast-derived proteins include and that circulate in the same fluid (130–132). Therefore, glycosaminoglycans, which are attached to one of two small disruption of the osteocyte network is likely to increase core proteins: PGI (or biglycan) and decorin; the latter has bone fragility. been implicated in the regulation of collagen fibrillogenesis. A number of other minor proteins such as osteopontin, bone C. Lining cells sialoprotein, fibronectin, vitronectin, and thrombospondin serve as attachment factors that interact with integrins. The surface of normal quiescent bone (i.e., bone that is In addition to being the cells that produce the osteoid not undergoing remodeling) is covered by a 1–2-␮m thick 122 MANOLAGAS Vol. 21, No. 2

FIG. 2. Functional syncytium compris- ing osteocytes, osteoblasts, bone marrow stromal cells, and endothelial cells. [Adapted from G. Marotti and reproduced with the permission of the Editor of Journal of Clinical Investigation 104: 1363–1374, 1999 (219).

layer of unmineralized collagen matrix on top of which vacuolar Hϩ-ATPase) located in the ruffled border mem- there is a layer of flat and elongated cells. These cells are brane. The protein components of the matrix, mainly colla- called lining cells and are descendents of osteoblasts (13). gen, are degraded by matrix metalloproteinases, and ca- Conversion of osteoblasts to lining cells represents one of thepsins K, B, and L are secreted by the osteoclast into the the fates of osteoblasts that have completed their bone area of bone resorption (134). The degraded bone matrix forming function; another being entombment into the ma- components are endocytosed along the ruffled border within trix as osteocytes. Osteoclasts cannot attach to the unmin- the resorption lacunae and then transcytosed to the mem- eralized collagenous layer that covers the surface of nor- brane area opposite the bone, where they are released (135, mal bone. Therefore, other cells, perhaps the lining cells, 136). Another feature of osteoclasts is the presence of high secrete collagenase, which removes this matrix before os- amounts of the phosphohydrolase enzyme, tartrate-resistant teoclasts can attach to bone. It has been proposed that acid phosphatase (TRAPase). This feature is commonly used targeting of osteoclast precursors to a specific location on for the detection of osteoclasts in bone specimens (137). Mice bone depends on a “homing” signal given by lining cells; deficient in TRAPase exhibit a mild osteopetrotic phenotype and that lining cells are instructed to do so by osteocytes, (due to an intrinsic defect of osteoclastic resorptive activity) the only bone cells that can sense the need for remodeling and defective mineralization of the cartilage in developing at a specific time and place (133). bones (138).

D. Osteoclasts VII. Death of Bone Cells by Apoptosis Mature osteoclasts are usually large (50 to 100 ␮m diameter) multinucleated cells with abundant mitochondria, nu- The average lifespan of human osteoclasts is about 2 merous lysosomes, and free ribosomes. Their most remark- weeks, while the average lifespan of osteoblasts is 3 able morphological feature is the ruffled border, a complex months (Table 1). After osteoclasts have eroded to a par- system of finger-shaped projections of the membrane, the ticular distance, either from the central axis in cortical bone function of which is to mediate the resorption of the calcified or to a particular depth from the surface in cancellous bone matrix (17, 123). This structure is completely sur- bone, they die and are quickly removed by phagocytes rounded by another specialized area, called the clear zone. (139). The majority (65%) of the osteoblasts that originally The cytoplasm in the clear zone area has a uniform appear- assembled at the remodeling site also die (140). The re- ance and contains bundles of actin-like filaments. The clear maining are converted to lining cells that cover quiescent zone delineates the area of attachment of the osteoclast to the bone surfaces or are entombed within the mineralized bone surface and seals off a distinct area of the bone surface matrix as osteocytes (Fig. 3A). Both osteoclasts and osteo- that lies immediately underneath the osteoclast and which blasts die by apoptosis, or programmed cell death, a pro- eventually will be excavated. The ability of the clear zone to cess common to several regenerating tissues (141). As in seal off this area of bone surface allows the formation of a other tissues, bone cells undergoing apoptosis are recog- microenvironment suitable for the operation of the resorp- nized by condensation of chromatin, the degradation of tive apparatus. DNA into oligonucleosome-sized fragments, and the for- The mineral component of the matrix is dissolved in the mation of plasma and nuclear membrane blebs (Fig. 4). acidic environment of the resorption site, which is created by Eventually the cell breaks apart to form so-called apoptotic the action of an ATP-driven proton pump (the so-called bodies. Osteoblast apoptosis explains the fact that 50–70% April, 2000 BIRTH AND DEATH OF BONE CELLS 123

FIG. 3. Osteoblast apoptosis and its implications. A, The average life span of a matrix forming osteoblast (ϳ200 h in the mouse) is indicated by the continuous line. The process of apoptosis represents only a small fraction of this time period. The alternative two fates of osteoblasts are to become lining cells or osteocytes. The fraction of osteoblasts that undergo apoptosis in vivo (fApoptosis) can be estimated from a bone biopsy specimen. The duration of the apoptosis phase that can be observed in the specimen (tApop) depends on the sensitivity of the detection method. For example, in the case of the TUNEL technique (without CuSO4 enhancement), the TUNEL-labeled phase of apoptosis is estimated to be approximately 2 h. In a steady state, the fraction of cells at a particular stage is the same as the corresponding fraction of time spent in that stage. Assuming an apoptosis detection time of 2 h and a 200-h life span, a prevalence of TUNEL positive osteoblasts in the biopsy of 0.005 indicates that half of the osteoblasts die by apoptosis. B, A change in the timing and extent of osteoblast apoptosis (fApoptosis) from 50% to zero should increase the number of osteoblasts present at the site of bone formation and thereby the work output, i.e., the amount of bone formed by a given team of matrix-forming osteoblasts. It will also lead to an increase in the density of osteocyte apoptosis, as illustrated by the example shown. of the osteoblasts initially present at the remodeling site of The same growth factors and cytokines that stimulate os- human bone cannot be accounted for after enumeration of teoclast and osteoblast development can also influence their lining cells and osteocytes (142). Moreover, the frequency apoptosis. For example, TGF␤ promotes osteoclast apoptosis of osteoblast apoptosis in vivo is such that changes in its while it inhibits osteoblast apoptosis. IL-6 type cytokines timing and extent could have a significant impact in the have antiapoptotic effects on animal and human osteoblastic number of osteoblasts present at the site of bone formation cells (and at least in vitro they antagonize proapoptotic effects (130). Osteocytes are long-lived but not immortal cells; of glucocorticoids) as well as on osteoclasts and their pro- some die by apoptosis (132, 143, 144). Osteocyte apoptosis genitors (54, 140, 145, 146). could be of importance to the origination and/or progression of the BMU. Indeed, osteocytes are the only cells in bone that can sense the need for remodeling at a specific VIII. Regulation of Bone Cell Proliferation and time and place. Moreover, osteocytes are in direct physical Activity contact with lining cells on the bone surface, and targeting of osteoclast precursors to a specific location on bone A large body of literature suggests that growth factors, depends on a “homing” signal given by lining cells (133). cytokines, hormones, and drugs regulate the proliferation of 124 MANOLAGAS Vol. 21, No. 2

FIG. 4. Two osteoblasts undergoing apoptosis in a section of murine cancellous bone (TUNEL staining with toluidine blue counterstain, ϫ630). Apoptotic osteoblasts (shown in brown) are adjacent to an intact osteoblast (shown in blue), on the surface of a trabecula occupying the right lower portion of the picture in which two intact (blue stained) osteocytes are also seen. Apoptotic osteoblasts display nuclear condensation and fragmentation. [Photomicrograph provided by Robert S. Weinstein, M.D., University of Arkansas for Medical Sci- ences.]

committed cells or the biosynthetic and functional activity of esis, or osteoclastogenesis, as used in this article, implicitly the differentiated osteoblasts and osteoclasts. Hence, in ad- combine commitment and amplification. dition to cell number, alterations in the functional activity of The in vivo relevance of numerous reports of in vitro ob- individual cells, i.e., cell vigor, may contribute to changes in servations of changes in the biosynthetic activity of osteo- the rate of bone resorption and formation. However, because blastic cells, e.g., changes in the level of expression of osteo- of the inherent difficulty in demonstrating changes in indi- calcin or alkaline phosphatase in response to a given agent, vidual cell vigor in vivo, the vast majority of such literature is also a matter of conjecture. Most likely, given the nature and its conclusions rely heavily, if not exclusively, on in vitro of commonly used in vitro cell models which, by and large, experimentation. A detailed discussion of this work is be- represent preterminally differentiated cells, such observa- yond the scope of this review, and the reader is referred to tions might be more relevant to postcommitment differen- other articles (30, 147–151). Nonetheless, some general as- tiation events, than to altered activity of the fully differen- pects merit discussion here, as they are important for putting tiated cell. Moreover, even if some agents can alter the the significance of birth rate and apoptosis into a broader function of terminally differentiated cells in short-term cul- perspective. tures in vitro, heretofore there is no evidence that short-term In regenerating tissues, the initial commitment of a stem change in the rate of collagen production or bone matrix cell progeny is followed by amplification with several or digestion, for example, are ultimately translated into differ- many rounds of cell division. In most tissues, division of stem ences in the amounts of bone matrix formed or resorbed. cells is infrequent, and almost all of the divisions that pro- In the bone literature there is considerable ambiguity duce the final population of differentiated cells occur in the when using the term “activation.” It is important that one so-called transit compartment (152). This notion is obscured distinguishes between activation as a switch from an off state by the frequent practice of showing linear diagrams repre- to an on state, and activation as modulation of activity of an senting the transition from one cell type to another and already active cell. Morphological evidence summarized ignoring completely the amplification during the transition. elsewhere does not support the notion that completely in- In general, terminally differentiated cells do not divide, and active osteoclasts are waiting for a stimulus to make them an osteoblast, defined as a cell making bone matrix, and active (117). However, this evidence does not address the osteoclast, as a cell resorbing bone, are in this category. issue of whether there are variations in the rate of bone Therefore, the in vivo relevance of much of the in vitro evi- matrix dissolution by individual osteoclasts after they started dence on the regulation of osteoblastic or osteoclastic cell work. proliferation, using established cell lines or primary cultures Studies with the widely used bone slice pit bioassay have of isolated cells, must be largely confined to changes in this shown that several regulatory factors can cause a decrease or transit compartment. Irrespective of whether a given regu- increase in the resorptive ability of individual osteoclasts latory factor, be that a cytokine or a hormone, influences the (153–155). However, it is not clear to what extent these ob- initial commitment of a stem cell, or the subsequent ampli- servations reflect a change in cell vigor as opposed to a fication of its progeny, or both, the end result is a change in change in the precariously short lifespan of osteoclasts in the rate of production and therefore the number of cells vitro. In estrogen deficiency, individual osteoclasts are seem- available for the execution of the biological task. For the sake ingly more “active” as they dig deeper resorption cavities of simplicity, the terms birth, rate of birth, osteoblastogen- often leading to trabecular perforation, but it has been con- April, 2000 BIRTH AND DEATH OF BONE CELLS 125 vincingly argued that this is due to delayed apoptosis (133). IX. Pathogenesis Of Osteoporosis In Paget’s disease osteoclasts are certainly far more aggres- From the brief discussion of the principles of physiological sive than normal, perhaps as a result of their uniquely large bone regeneration and the role of osteoblasts and osteoclasts size and number of nuclei (156). Today, the most compelling in the process, it is obvious that the rate of supply of new evidence in support of the notion that the vigor of individual osteoblasts and osteoclasts and the timing of the death of osteoclasts may not always be maximal comes from in vitro these cells by apoptosis are critical determinants of the ini- as well as in vivo experiments with soluble RANK-ligand tiation of new BMUs and/or extension or shortening of the (157). Specifically, it has been found that RANK-ligand acts lifetime of existing ones. Recent advances in our understand- on mature rat osteoclasts in vitro to stimulate more frequent ing of the pathogenesis of the various forms of osteoporosis cycles of resorption and induce rearrangements of the cy- have confirmed this truism by revealing that over- or un- toskeleton. Moreover, intravenous administration of RANK- dersupply of these cells relative to the need for remodeling ligand to mice elevates the circulating concentration of are the fundamental problems in all these conditions (160) ionized calcium within 1 h. RANK-ligand has potent anti- (Table 2). apoptotic effects on cultured osteoclasts (Ref. 158 and Wil- liam Boyle, personal communication). Therefore, definitive conclusions regarding RANK-ligand’s ability to modulate A. Sex steroid deficiency osteoclast vigor will have to await dissection of the contribution of its effects on osteoclast survival in vivo. Similar to The mechanism of action of sex steroids on the skeleton is osteoclasts, the bone-forming ability of osteoblasts may not not fully understood. At menopause (or after castration in be always maximal, as suggested by the evidence that PTH men), the rate of bone remodeling increases precipitously. can rapidly enhance bone formation when administered by This fact may be explained by evidence, derived primarily subcutaneous injections to rats (159). from studies in mice, that loss of sex steroids up-regulates the To conclude this section, it is intuitive that the amount of formation of osteoclasts and osteoblasts in the marrow by bone resorbed or formed by a team of osteoclasts and os- up-regulating the production and action of cytokines that are teoblasts should be a function of the total cell number as well responsible for osteoclastogenesis and osteoblastogenesis as the vigor of individual cells. However, whereas cell num- (21, 161, 162). Indeed, both estrogen and androgen suppress ber can be quantified on bone sections from animals and the production of IL-6, as well as the expression of the two humans with conventional histomorphometric techniques, subunits of the IL-6 receptor, IL-6R␣ and gp130, in cells of the quantification of individual cell vigor cannot. This situation bone marrow stromal/osteoblastic lineage (40, 163). Sup- makes it difficult to judge at present whether cell vigor is or pression of IL-6 production by estrogen or selective estrogen is not a critical component in the pathogenesis of abnormal receptor modulators (SERMs), such as raloxifene, does not skeletal regeneration in common acquired metabolic bone require direct binding of the estrogen receptor to DNA. In- diseases such as postmenopausal, senile, or steroid-induced stead, it is due to protein-protein interaction between the osteoporosis. For this reason and space limitations, changes estrogen receptor and transcription factors such as NF-K␤ in bone cell vigor or other potential mechanisms of osteo- and C/EBP. This mechanism provides a model that best fits porosis resulting from changes in extraskeletal tissues, for current understanding of the molecular pharmacology of example altered calcium absorption or excretion, will not be estrogen and SERMs (164). Consistent with the suppressive discussed in the following section. This also reflects the au- effect of sex steroids on IL-6 and its receptor, several, albeit thor’s intention to focus on the dynamics of bone cell num- not all, studies have shown that the level of expression of ber, rather than a dismissal of other mechanisms. IL-6, as well as IL-6R␣ and gp130, is elevated in estrogen-

TABLE 2. Cellular changes and their culprits in the three most common causes of bone loss

Cellular changes Probable culprits Sex steroid deficiency 1Osteoblastogenesis Increased IL-6; TNF; IL-1RI/IL-RII 1Osteoclastogenesisa MCSF; decreased TGF␤; OPG 1Lifespan of osteoclasts Loss of pro- and antiapoptotic effects of sex steroids, respectively 2Lifespan of osteoblasts 2Lifespan of osteocytes Senescence 2Osteoblastogenesisb Increased PPAR␥2, PGJ2, noggin; Deceased IL-11, IGFs 2Osteoclastogenesis 1Adipogenesis 2Lifespan of osteocytes Glucocorticoid excess 2Osteoblastogenesis Decreased Cbfal and TGF␤ R1; and BMP-2 and IGF1 action 2Osteoclastogenesis 1Adipogenesis Increased PPAR␥2 1Lifespan of osteoclastsc 2Lifespan of osteoblasts Decreased Bcl-2/BAX ratio 2Lifespan of osteocytes a Oversupply of osteoclasts relative to the need for remodeling. b Undersupply of osteoblasts relative to the need for cavity repair. c Osteoclast numbers may transiently increase in the earlier stages of steroid therapy, without an increase in osteoclastogenesis, indicating increased lifespan. 126 MANOLAGAS Vol. 21, No. 2 deficient mice and rats as well as in humans, in the bone are more widely separated and less well connected. An marrow and in the peripheral blood (165–168). Furthermore, equivalent amount of cancellous bone distributed as widely neutralization of IL-6 with antibodies or knockout of the IL-6 separated, disconnected, thick trabeculae is biomechanically gene in mice prevents the expected cellular changes in the less competent than when arranged as more numerous, con- marrow and in trabecular bone sections and protects the mice nected, thin trabeculae. Concurrent loss of cortical bone oc- from bone loss after loss of sex steroids (51, 67). Consistent curs by enlargement and coalescence of subendocortical with its pathogenetic role in the bone loss caused by loss of spaces, a process due to deeper penetration of endocortical sex steroids, IL-6 seems to play a similar role in several other osteoclasts. conditions associated with increased bone resorption as ev- This deeper erosion can be now explained by evidence that idenced by increased local or systemic production of IL-6 and estrogen acts on mature osteoclasts to promote their apo- the IL-6 receptor in patients with multiple myeloma, Paget’s ptosis; consequently, loss of estrogen leads to prolongation disease, rheumatoid arthritis, Gorham-Stout or disappearing of the lifespan of osteoclasts (133). Specifically, estrogen pro- bone disease, hyperthyroidism, primary and secondary hy- motes osteoclast apoptosis in vitro and in vivo by 2- to 3-fold, perparathyroidism, as well as McCune Albright Syndrome an effect seemingly mediated by TGF␤ (139). In direct con- (66, 68, 169–174). trast to their proapoptotic effects on osteoclasts, estrogen (as In line with the fact that loss of sex steroids increases the well as androgen) exerts antiapoptotic effects on osteoblasts rate of bone remodeling, in addition to up-regulating oste- and osteocytes; consequently, loss of estrogen or androgen oclastogenesis, loss of sex steroids increases the number of leads to shorter lifespan of osteoblasts and osteocytes (184). osteoblast progenitors in the murine bone marrow. These Extension of the working life of the bone-resorbing cells and changes are temporally associated with increased bone for- simultaneous shortening of the working life of the bone- mation and parallel the increased osteoclastogenesis and forming cells, can explain the imbalance between bone re- bone resorption (175). As IL-6 type cytokines can stimulate sorption and formation that ensues after loss of sex steroids. osteoblast development and differentiation (54, 55, 146), in- Furthermore, the increase in osteocyte apoptosis could fur- creased sensitivity to IL-6 and other members of this cyto- ther weaken the skeleton by impairment of the osteocyte- kine’s family may account also for the increased osteoblast canalicular mechanosensory network. The increase in bone formation that follows the loss of gonadal function. In view remodeling that occurs with estrogen deficiency would of the fact that mesenchymal cell differentiation and oste- partly replace some of the nonviable osteocytes in cancellous oclastogenesis are tightly linked, stimulation of mesenchy- bone, but cortical apoptotic osteocytes might accumulate mal cell differentiation toward the osteoblastic lineage after because of their anatomic isolation from scavenger cells and sex steroid loss may be the first event that ensues after the the need for extensive degradation to small molecules to hormonal change, and increased osteoclastogenesis and dispose of the osteocytes through the narrow canaliculi. bone loss might be downstream consequences of this change Hence, the accumulation of apoptotic osteocytes caused by (106). loss of estrogen, or glucocorticoid excess (130), could increase In addition to IL-6, estrogen also suppress TNF and M-CSF bone fragility even before significant loss of bone mass, be- (176, 177), and estrogen loss may increase the sensitivity of cause of the impaired detection of microdamage and repair osteoclasts to IL-1 by increasing the ratio of the IL-1RI over of substandard bone. the IL-1 receptor antagonist (IL-RII) (178). As in the case of In conclusion, the increased rate of bone remodeling in IL-6, the effects of estrogen on TNF and M-CSF are mediated estrogen deficiency may be due to increased production of via protein-protein interactions between the estrogen recep- both osteoclasts and osteoblasts, and the imbalance between tor and other transcription factors. In agreement with the bone resorption and formation is due to an extension of the evidence that IL-1 and TNF play a role in the bone loss caused working lifespan of the osteoclast and shortening of the by loss of estrogen, administration of IL-1RA and/or a TNF working lifespan of the osteoblast. Moreover, a delay of soluble receptor ameliorates the bone loss caused by ovari- osteoclast apoptosis seems responsible for the deeper re- ectomy in rats and mice (179–181). Because of the interde- sorption cavities and thereby the trabecular perforation as- pendent nature of the production of IL-1, IL-6, and TNF, a sociated with estrogen deficiency. significant increase in one of them may amplify, in a cascade Clinical observations of decreased bone mass in a male fashion, the effect of the others (161). Interestingly, recent in with mutant estrogen receptor (185), and increased bone vitro studies with human osteoblastic cells indicate that OPG mass after treatment with estrogen in two males with P-450 production is stimulated by estrogen, suggesting that this aromatase deficiency (186, 187), have raised the possibility cytokine may also play an important role in the antioste- that estrogen derived by peripheral aromatization of andro- oclastogenic (antiresorptive) action of estrogen on bone (182). gens is critical for the maintenance of bone mass in men as Increased remodeling, resulting from up-regulation of os- well as in women (188). However, in all three cases, the teoblastogenesis and osteoclastogenesis, alone can cause a decreased bone mass in young males with estrogen defi- transient acceleration of bone mineral loss because bone re- ciency in the face of androgen sufficiency could be due to sorption is faster than bone formation, and new bone is less failure in achieving peak bone mass from defects occurring dense than older bone. However, in addition to increased during development or growth, not to loss of bone mass, as bone remodeling, loss of sex steroids leads to a qualitative it is the case with common forms of osteoporosis. In addition, abnormality: osteoclasts erode deeper than normal cavities individuals with complete androgen insensitivity, due to (133, 183). In this manner, sex steroid deficiency leads to the mutations in the androgen receptor gene on the X chromo- removal of some cancellous elements entirely; the remainder some and increased testosterone and estrogen production, April, 2000 BIRTH AND DEATH OF BONE CELLS 127 have decreased bone mass, in spite of the elevated estrogen HODE, are endogenous ligands and activators of PPAR␥ levels (189). Moreover, androgens, including nonaromatiz- (201), and PPAR␥ promotes monocyte/macrophage differ- able ones, have identical effects to those of estrogen on the entiation and uptake of oxidized LDL (202). Taken together biosynthetic activity and the birth as well as the death of bone with these findings, the evidence that activated PPAR␥2 cells in vitro and in vivo, at least in rodents (67, 106, 190). It promotes adipocyte differentiation at the expense of osteo- is therefore more likely that both estrogen and androgen are blastogenesis in the bone marrow by suppressing Cbfa1 (94) important for the maintenance of bone mass in the adult male suggests a mechanistic link among dietary fat/lipoproteins, skeleton. bone marrow stromal cell differentiation, osteoporosis, and atherogenesis. In support of the existence of such a link, ␥ B. Senescence activation of PPAR by thiazolidinediones or oxidatively modified LDL inhibits osteoblastogenesis of bone marrow- The amount of bone formed during each remodeling cycle derived stromal cells in vitro (94, 203, 204). Moreover, high decreases with age in both sexes. This is indicated by a fatty acid content in rabbit serum or high fat diet of mice for consistent histological feature of the osteopenia that occurs 4 months decreases osteogenic cell differentiation in ex vivo during aging, namely a decrease in wall thickness, especially bone marrow cell cultures (86, 204). These new advances may in trabecular bone (191–193). Wall thickness is a measure of explain clinical observations that atherosclerosis and osteo- the amount of bone formed in a remodeling packet of cells porosis coexist (205, 206). and is determined by the number or activity of osteoblasts at Quantitative trait loci (QTLs) analysis of osteopenia-asso- the remodeling site. ciated loci using closely related mouse strains have mapped Studies measuring bone turnover by histomorphometry five loci to regions of chromosomes 2, 7, 11, and 16 (207). (194), or indirectly by circulating markers (195–197), have Association of these same loci with bone mineral density has suggested that in aging women, even in extreme old age, been reproduced in crosses of different recombinant-inbred bone turnover is most likely increased by secondary hyper- mouse strains (208, 209). Such recurrent appearance of QTL, parathyroidism or by the continuing effect of estrogen de- especially in crosses involving distantly related strains, im- ficiency. Increased turnover and reduced wall thickness are plies that polymorphism at these loci may be favored by not inconsistent, as the former is the result of increased evolution and might underlie variation in peak bone density activation frequency, and the decreased wall thickness—an among humans. Intriguingly, of the more than 12 genes index of decreased bone formation by osteoblasts—in senes- affecting bone homeostasis that were localized near these cence is local and relative to the demand created by resorp- QTLs, 2 are prostaglandin synthases, a third is the BMP-2/4 tion. antagonist noggin, a fourth is the proapoptotic protein bax, Changes in the birth of bone cells in the bone marrow and the fifth is IL-11. Hence, the transcription factor PPAR␥2 provide a mechanistic explanation for the contribution of and its ligand, PGJ2, noggin, and IL-11 are potentially re- senescence to bone loss, independently from sex steroid de- sponsible for the decreased osteoblastogenesis with advanc- ficiency. Specifically, using SAMP6 mice, a murine model of ing age. This contention is supported by the evidence that the age-related osteopenia (but sufficient in sex steroids and with reciprocal relationship between decreased osteoblastogen- intact reproductive function), a tight association among re- esis and increased adipogenesis in the SAMP6 mouse may be duced number of osteoblast progenitors, decreased bone for- explained by a change in the expression of PPAR␥ or its mation, and decreased bone mass has been established (105). ligands in early mesenchymal progenitors; that BMP-2/4, in Decreased osteoblastogenesis with advancing age has been balance with noggin, may determine the tonic baseline con- confirmed in the human bone marrow (198, 199). Impor- trol of the rate of osteoblastogenesis; and that IL-11 is a potent tantly, the decrease in osteoblastogenesis is accompanied by inhibitor of adipogenesis, which stimulates osteoblast dif- increased adipogenesis and myelopoiesis, as well as de- ferentiation and the expression of which is reduced in creased osteoclastogenesis, the latter most likely caused by a SAMP6 mice. In addition to these factors, growth factors such reduction in the stromal/osteoblastic cells that support os- as IGFs have also been implicated in the bone loss associated teoclast formation (105, 200). This suggests that in aging there with senescence (210, 211). Irrespective of the identity of the must be changes in the expression of genes that favor the precise mediator, the reciprocal change between adipogen- differentiation of multipotent mesenchymal stem cells to- esis and osteoblastogenesis can explain the association of ward adipocytes at the expense of osteoblasts. The evidence decreased relative bone formation and the resulting osteope- that PPAR␥2 induces the terminal differentiation of marrow nia with the increased adiposity of the marrow seen with cells with both osteoblastic and adipocytic characteristics to advancing age in animals and humans (105, 142, 212–215). adipocytes, and simultaneously suppresses Cbfa1 expression and terminal differentiation to osteoblasts (94), raises the C. Glucocorticoid excess possibility that increased expression of PPAR␥2 or its ligands, e.g., PGJ2, may be some of the culprits responsible for The cardinal histological features of glucocorticoid-induced the reciprocal change between adipogenesis and osteoblas- osteoporosis are decreased bone formation rate, decreased wall togenesis with advancing age (Table 2). thickness of trabeculae (a strong indication of decreased work Uptake of oxidized low-density lipoproteins (LDL) play an output by osteoblasts), and in situ death of portions of bone. important role in foam cell formation and the pathogenesis Increased bone resorption, decreased osteoblast proliferation of atherosclerosis. Two of the major components of oxidized and biosynthetic activity, and sex-steroid deficiency, as well as LDL, 9-hydroxy-9,11-octadecadienoic acid (HODE) and 13- hyperparathyroidism resulting from decreased intestinal cal- 128 MANOLAGAS Vol. 21, No. 2 cium absorption and hypercalciuria due to defective vitamin D from patients with glucocorticoid-induced osteoporosis ex- metabolism, have all been proposed as mechanisms for the loss hibit abundant apoptotic osteocytes adjacent to the subchon- of bone that ensues with glucocorticoid excess (216). dral fracture crescent (221). Glucocorticoid-induced osteo- The decreased bone formation and osteonecrosis can now cyte apoptosis, a cumulative and unrepairable defect, could be explained by evidence that glucocorticoid excess has a uniquely disrupt the proposed mechanosensory role of the suppressive effect on osteoblastogenesis in the bone marrow osteocyte network and thus promote collapse of the femoral and also promotes the apoptosis of osteoblasts and osteo- head. cytes (118). Indeed, mice receiving glucocorticoids for 4 At this time, the mediators of the cellular changes caused weeks, a period equivalent to ϳ3–4 yr in humans, exhibit by glucocorticoid excess are only a matter of conjecture. decreased bone mineral density associated with a decrease in Nonetheless, glucocorticoids directly suppress BMP-2 and the number of osteoblast, as well as osteoclast, progenitors in Cbfa1-2—two critical factors for osteoblastogenesis—and the bone marrow and a dramatic reduction in cancellous may also decrease the production of IGFs while they stim- bone area and in trabecular width compared with placebo ulate the transcriptional activity of PPAR␥2 (222–225) (Table controls. These changes are associated with a significant re- 2). duction in osteoid area and a decrease in the rates of mineral apposition and bone formation. More strikingly, glucocorticoid administration to mice causes a 3-fold increase in the X. Pharmacotherapeutic Implications of Osteoblast prevalence of osteoblast apoptosis in vertebrae and induced and Osteocyte Apoptosis apoptosis in 28% of the osteocytes in metaphyseal cortical Estrogen replacement therapy (ERT), various bisphospho- bone. Nevertheless, even though there is a significant cor- nates (e.g., alendronate), the SERM raloxifene, calcitonin, and relation between the severity of the bone loss and the extent sodium fluoride, as well as calcium and vitamin D, are ap- of reduction in bone formation, some of the bone loss may proved modalities for the prevention and treatment of bone be due to an early increase in bone resorption as evidenced loss, irrespective of its cause. Decreased osteoclast progenitor by an early increase in osteoclast perimeter of vertebral can- development and/or decreased osteoclast recruitment and cellous bone after 7 days of steroid treatment. In vivo studies promotion of apoptosis of mature osteoclasts leading to a with mice from this author’s group show that at this early slowing of the rate of bone remodeling are thought to be the time point (7 days after glucocorticoid administration) os- main mechanisms of the so-called “antiresorptive” agents teoclastogenesis in ex vivo bone marrow cultures is decreased estrogen, bisphosphonates, SERMs, and calcitonin. Sodium by half, while the number of osteoclasts in bone sections fluoride has anabolic properties, but its therapeutic range is doubles (Robert Weinstein, personal communication), sug- very narrow. Calcium and vitamin D are rarely sufficient on gesting that an early effect of glucocorticoid excess might be their own, but they are considered a very useful supplemen- increased osteoclast survival. In vitro studies by others, on the tation in any regimen for osteoporosis. other hand, have shown that glucocorticoids inhibit OPG and concurrently stimulate the expression of RANK-ligand in A. Intermittent PTH administration human osteoblastic, primary, and immortalized bone marrow stromal cells (217). Taken together, these lines of evi- The ideal therapy for osteoporosis, especially in elderly dence suggest that the initial rapid phase of bone loss with patients who already have advanced bone loss, would be an glucocorticoid treatment could be due to an extension of the anabolic agent that will increase bone mass by rebuilding lifespan of preexisting osteoclasts, mediated by RANK- bone. It is well established that daily injections of low doses ligand (117). of PTH—an agent better known for its role in calcium ho- The same histomorphometric changes that have been meostasis—increases bone mass in animals and humans found in mice after a 4-week treatment with steroids have (226–231) as does the PTH-related protein (PTHrP), the only been confirmed in biopsies from patients receiving long-term other known ligand of the PTH receptor (232). Indeed, al- glucocorticoid therapy. Moreover, as in mice, an increase in though constant, high levels of PTH cause increased bone osteoblast and osteocyte apoptosis is found in human biop- resorption and osteitis fibrosa cystica, low and intermittent sies. Compared with osteoblast apoptosis, apoptotic osteo- doses of PTH, too small to affect serum calcium concentra- cytes are far more prevalent, at least in metaphyseal cortices, tions, promote bone formation and increase bone mineral probably because of the anatomical isolation of osteocytes density at the lumbar spine and hip. This so-called anabolic from scavenger cells. Consistent with these findings, glu- effect can be now explained by evidence that PTH increases cocorticoids promote osteoblast and osteocyte apoptosis in the life span of mature osteoblasts in vivo by reducing the vitro (218, 219). Decreased production of osteoclasts can ex- prevalence of their apoptosis from 1.7–2.2% to as little as plain the reduction in bone turnover with chronic glucocor- 0.1–0.4% rather than by affecting the generation of new os- ticoid excess, whereas decreased production and apoptosis teoblasts (218). The antiapoptotic effect of PTH in mice was of osteoblasts can explain the decline in bone formation and sufficient to account for the increase in bone mass and was trabecular width. Accumulation of apoptotic osteocytes may confirmed in vitro using rodent and human osteoblasts and also explain the so-called “osteonecrosis,” also known as osteocytes. Like PTH, PGE inhibits periosteal cell apoptosis aseptic or avascular necrosis, another manifestation of ste- via cAMP-dependent stimulation of sphingosine kinase roid-induced osteoporosis that causes collapse of the femoral (233). Interestingly, whereas PTH inhibits apoptosis in cells head in as many as 25% of patients (220). This contention is overexpressing Gs, an activator of adenylate cyclase, PTH supported by evidence that whole femoral heads obtained stimulates apoptosis via G protein-coupled receptors in cells April, 2000 BIRTH AND DEATH OF BONE CELLS 129 overexpressing Gq (an activator of JNK and calcium signal- also have a positive effect on bone formation. An explanation ing), suggesting that the antiapoptotic effects of PTH are for this evidence is now provided by studies demonstrating mediated by signals transduced through the Gs pathway that bisphosphonates such as etidronate, alendronate, pam- (234). idronate, olpadronate, or amino-olpadronate (IG9402, a Osteocytes in the newly made lamellar cancellous bone bisphosphonate that lacks antiresorptive activity), as well as in the mice receiving daily PTH injections were closer calcitonin have antiapoptotic effects on osteoblasts and os- together and more numerous than those found in the teocytes (219). These effects are associated with a rapid in- animals receiving vehicle alone (218). The closely spaced, crease in the phosphorylated fraction of extracellular regu- more numerous osteocytes are the predictable conse- lated signal kinases (ERKs) and are blocked by specific quence of protecting osteoblasts from apoptosis (Fig. 3B). inhibitors of ERK activation. In agreement with the in vitro The antiapoptotic effect of PTH on osteoblasts as well as results, alendronate abolishes the increase in the prevalence osteocytes has been confirmed in vitro using primary bone of vertebral, cancellous bone osteocyte and osteoblasts ap- cell cultures and established cell lines. Elucidation of this optosis induced by administration of prednisolone in mice. mechanism provides for the first time proof that inhibition These findings raise for the first time the possibility that of osteoblast apoptosis may represent a novel therapeutic increased survival of osteoblasts and osteocytes may both strategy for augmenting bone mass. Be that as it may, contribute to the efficacy of bisphosphonates and calcitonin several alternative mechanisms, including activation of in the management of disease states due to loss of bone. lining cells, have been proposed, and they may also con- If both “antiresorptive” and “anabolic” agents [e.g., in- tribute to the anabolic effect of PTH (235–237). Nonethe- termittent PTH] prevent osteoblast and osteocyte apopto- less, lining cells cover at least 3 times more surface than sis, why is increased formation so much more apparent in osteoblasts. Therefore, conversion of lining cells to bone- the case of the “anabolic” agents? The discussion of phys- forming osteoblasts alone would be insufficient to cover iological bone regeneration in the beginning of this review the increased cancellous bone area observed in rats and to article provides an answer. Bone formation occurs only on account for the expanded bone perimeter and the in- sites of previous osteoclastic bone resorption, i.e., on sites creased osteocyte number and density observed with PTH undergoing remodeling. Each remodeling cycle is a trans- treatment in mice (218). action that, once consummated, is irrevocable. Therefore, Daily subcutaneous injections of PTH are safe and effec- agents with antiapoptotic properties that do not have an- tive in the treatment of patients with corticosteroid-induced tiresorptive/antiremodeling properties, i.e., they do not osteoporosis (230). The elucidation of the importance of os- decrease the number of remodeling units, are expected to teoblast and osteocyte apoptosis in the mechanism of glu- rebuild more bone and therefore increase the overall bone cocorticoid-induced osteoporosis, and the elucidation of the mass, because of the greater number of profitable trans- importance of preventing apoptosis in the anabolic effects of actions. Hence, by decreasing the prevalence of osteoblast PTH on bone, readily explain how PTH can be such an apoptosis, agents with pure antiapoptotic properties, such effective therapy in this condition. Hence, PTH and perhaps as intermittent PTH, can expand the pool of mature os- future PTH mimetics represent, for the first time, pathophys- teoblasts at sites of new bone formation and allow these iology-based, i.e., rational as opposed to empirical, pharma- cells more time to make bone, to a much greater degree cotherapies for osteopenias, in particular, those in which than the antiresorptive agents that also slow remodeling. osteoblast progenitor formation is suppressed. In any case, However, in the case of either class of agents, upholding future studies to assess the antifracture efficacy of these the osteocyte-canalicular network by preventing osteocyte compounds will be needed before their effectiveness for the apoptosis, should contribute to antifracture efficacy, over management of osteoporosis can be established. and above that resulting from their effects on bone mass (Fig. 5). Therefore, the distinction between antiresorptive B. Bisphosphonates and calcitonin and anabolic agents may be more apparent than real when it comes to antifracture efficacy. Bisphosphonates, stable analogs of pyrophosphate, and calcitonin are potent inhibitors of bone resorption and ef- C. Novel pharmacotherapeutic strategies fective therapies for the management of osteoporosis and other diseases characterized by bone loss (238, 239). The main Based on the understanding of the role of growth factors mechanism of the antiresorptive actions of these agents is on osteoblast development, proliferation, and differentia- decreased development of osteoclast progenitors, decreased tion, several of them (e.g., GH, the insulin-like growth factors osteoclast recruitment, and promotion of apoptosis of mature I and II, TGF␤, BMPs, and FGF) have been advocated as osteoclasts leading to a slowing rate of bone remodeling (133, potential future therapeutic agents for the management of 240–242). Nonetheless, the antifracture efficacy of these bone loss (247, 248). However, with the exception of BMPs, agents is disproportional to their effect on bone mass (243), which may be of value in local augmentation of bone mass suggesting an additional effect on bone strength unrelated to and acceleration of fracture healing, none of them has been effects on bone resorption. Moreover, long-term treatment of shown to be efficacious (let alone safe or convenient and human and nonhuman primates with bisphosphonates has practical) for the management of common metabolic bone been shown to increase wall thickness, an index of increased disorders such as osteoporosis. The recent elucidation of the osteoblast numbers or activity (244–246), raising the possi- mechanism of the anabolic effects of PTH, and specifically bility that they may not only inhibit bone resorption, but may the demonstration of increased work output of a cell pop- 130 MANOLAGAS Vol. 21, No. 2

FIG. 5. Hypothetical model of the implications of the effects of antiresorptive (i.e., antiremodeling) vs. non-antiresorptive agents on prolonging the life span of mature osteoblasts and osteocytes. For explanation, please see text.

ulation by suppressing apoptosis to augment tissue mass, a potent neuroprotective compound (264–266), and does ex- points to an entirely new avenue for future drug discovery. hibit potent antiapoptotic effects on osteoblasts and osteo- Indeed, in addition to PTH and PTH mimetics, which by cytes in vitro. In support of the hypothesis that ANGELS can virtue of their peptidic nature carry the inconvenience of be used as a novel, advantageous mode of therapy for the daily injections, one can for the first time envisage nonpep- augmentation of bone mass and/or fracture prevention in tide inhibitors of proapoptotic pathways in osteoblasts and diseases characterized by low bone mass and increased fra- osteocytes as therapeutic agents for osteopenias and espe- gility, preliminary evidence indicates that estratriene-3-ol cially those in which osteoblast progenitors are low, such as increases BMD and bone strength in both estrogen-replete age-related and glucocorticoid-induced osteoporosis. and estrogen-deficient mice (261). In view of this preclinical As discussed in Section VIII in this review, estrogen and finding and the evidence that androgen (190), as well as androgen deficiency increase osteoblast and osteocyte apo- estrogen, have antiapoptotic effects on osteoblasts and os- ptosis in humans, rats, and mice; and these changes have teocytes, one is encouraged to think that estrogenic, andro- been shown to be reversed by replacement therapy, at least genic, or even nonsteroidal compounds that can activate in mice (132, 144, 184, 190). In full agreement with these in antiapoptotic, but not antiremodeling, signals on osteoblasts vivo observations, 17␤-estradiol inhibits osteoblast and os- and osteocytes, are candidates for future osteoporosis treat- teocyte apoptosis in vitro. The antiapoptotic effect of 17␤- ments that, unlike existing ones that prevent or retard bone estradiol on osteoblasts and osteocytes require the presence loss, may augment bone mass. of the estrogen receptor-␣ or -␤ (249). Nonetheless, unlike the classical mechanism of estrogen receptor action that involves direct or indirect interaction with the transcriptional appa- XI. Summary and Conclusions ratus, the estrogen receptor-dependent antiapoptotic effect of 17␤-estradiol is due to rapid (within 5 min) phosphory- In 1995, it was proposed that “changes in the numbers of lation of ERKs (250). Moreover, the antiapoptotic effect of bone cells, rather than changes in the activity of individual 17␤-estradiol can be reproduced by 17␣-estradiol, a com- cells, form the pathogenetic basis of osteoporosis”; and that pound thought of as an inactive analog of 17␤-estradiol, as “excessive osteoclastogenesis and inadequate osteoblasto- well as a membrane-impermeable conjugate of 17␤-estradiol genesis are responsible for the mismatch between the for- ␤ with BSA (17 E2-BSA). Numerous effects of estrogen have mation and resorption of bone in postmenopausal and age- been observed over the last few years in a variety of cell related osteopenia” (21). Since then, this paradigm shift of types, including osteoblasts, the rapidity of which makes a thinking has led to important new discoveries that, along genomic mechanism of action unlikely (251–256). Many of with several other independent breakthroughs, refine the these rapid actions have been attributed to the ability of concept of “cell number” and broaden its relevance to the estrogen to act at the cell membrane on a membrane-asso- physiology and pathophysiology of bone at large. Moreover, ciated estrogen receptor (257–260). The antiapoptotic effects these discoveries provide a new landscape for critical re- of estrogen on osteoblasts and osteocytes fall into this cate- evaluation of our current thinking about therapeutic strate- gory of “nongenomic” actions. Based on this, the term “ac- gies for bone diseases. Indeed, it is now clear that bone cells tivators of non-genomic estrogen-like signaling” (ANGELS), must be continually replaced, and the number present de- has been coined for compounds that mimic the nongenomic pends not only on their birth rate, which reflects the fre- effects of estrogen, but have reduced classical estrogenic quency of cell division of the appropriate precursor cell, but actions (261). A paradigm of such agents is the synthetic also on the life span, which most likely reflects the timing of compound estratriene-3-ol, which has decreased transcrip- death by apoptosis. The process of replacement of osteoblasts tional activity as compared with 17␤-estradiol (262, 263), is and osteoclasts is tightly coordinated and orchestrated at the April, 2000 BIRTH AND DEATH OF BONE CELLS 131 early progenitor level. Changes in the birth rate and/or ap- 15. 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STADY II-International Symposium on Signal Transduction in Health and Disease September 12–15, 2000 Tel Aviv, Israel

For further information, contact: Professor Zvi Naor Department of Biochemistry Tel Aviv University Tel Aviv, Israel Telephone: 972-3-640-9032/641-7057 Fax: 972-3-640-6834 E-mail: [email protected] or [email protected] IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493

PERSPECTIVES

The Osteoclast Cytoskeleton: How Does It Work?

Steven L. Teitelbaum and Wei Zou Washington University School of Medicine, St. Louis, Missouri, USA

Abstract

The capacity of the osteoclast to resorb bone is distinctive, as is the cell’s appearance. Both characteristics reflect cytoskeletal organization that yields structures such as the sealing zone and ruffled border. The unique nature of these organelles and their dependence upon contact with bone have been appreciated for some time but insights into the mechanisms by which they are generated come from more recent studies. These insights include the role of integrins, particularly αvβ3, in cytoskeletal organization and the canonical signaling pathway they activate. Investigators now appreciate that the sealing zone isolates the resorptive microenvironment from the general extracellular space, permitting secretion of matrix- degrading molecules on the bone surface. Thus, the osteoclast is a secretory cell that depends upon polarization of exocytic vesicles to the bone-apposed plasma membrane into which they insert under the aegis of vesicle/membrane fusion proteins. This process focally expands and convolutes the plasmalemma included within the sealing zone, eventuating in formation of the ruffled border. Many of these events are now better understood and are the focus of this Perspective. IBMS BoneKEy. 2011 February;8(2):74-83. 2011 International Bone & Mineral Society

Isolating the Resorptive gasket-like, sealing zone. A single Microenvironment osteoclast, being a large cell, enjoys multiple contacts with bone and therefore generates Osteoclasts are polykaryons and members numerous sealing zones and resorptive of the macrophage lineage with the unique microenvironments. capacity to degrade the inorganic and organic matrices of bone. If excessive, the When most other cells attach to matrix they bone resorptive activity of osteoclasts generate focal adhesions. These stable causes osteoporosis. Conversely, the structures contain integrins and signaling osteoclast initiates remodeling that likely and cytoskeletal molecules that, upon removes structurally compromised bone, contact with matrix, promote formation of thereby maintaining mechanical integrity (1). actin stress fibers. Consistent with the lack Skeletal health, therefore, requires optimal of actin stress fibers in mammalian osteoclast function. osteoclasts, they lack focal adhesions but in their place develop podosomes (2;3). These Resorption is initiated by attachment of punctuate structures contain an F-actin core osteoclasts to bone. They then develop a and a peripheral “cloud” of a loose network compartment between their plasma of radial actin cables (4-7). In contrast to membrane and the bone surface into which focal adhesions, podosomes are transient the cells transport matrix-degrading but mediate substrate adhesion and thus molecules including H+ and Cl-, which, in formation of the resorptive concert, demineralize the target bone, and microenvironment. cathepsin K, which degrades the exposed collagen fibers and associated proteins. To Most past studies of osteoclast podosomes isolate this resorptive microenvironment utilized cells resident on plastic or glass. In from the general extracellular space, these circumstances, the punctuate osteoclasts reorganize their cytoskeleton to structures initially appear in clusters but generate an encompassing, actin-rich, ultimately coalesce, first into an

IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 intracytoplasmic actin ring and then a degrading bone and its absence indicates peripheral actin belt (4). The physiological the cell is not doing so. Reflecting multiple relevance of this observation was contacts with bone and attendant sealing challenged by the absence of apparent zones, ruffled borders are also numerous in podosomes or an actin belt in non-stressed a given osteoclast. osteoclasts on bone (4). While the relevance of the peripheral belt is not established, it is This complex enfolding of the plasma clear that, like the actin ring, the sealing membrane, unique to the osteoclast, abuts zone of bone-residing osteoclasts also and extends into the resorptive space. It is consists of podosome-containing structural surrounded by the sealing zone and is the units (8). venue by which the cell secretes matrix- degrading molecules on the bone surface Microtubules Are Important (17). It is therefore the resorptive organelle and is absent or deranged in many forms of Depending upon their state of acetylation, osteopetrosis. As osteoclast resorption microtubules in osteoclasts are transient or alternates with migration, the ruffled border polymerized and relatively stable (9-11). is a transient structure. Unlike actin ring formation by glass-residing cells, sealing zone generation in bone- The ruffled border, which forms only upon resorbing osteoclasts is characterized by contact with mineralized substrate, is microtubule acetylation, again illustrating the initiated by transport of cathepsin K – and/or influence of substrate on the cell’s H+ATPase – and Cl- channel-bearing cytoskeleton (10). vesicles to the bone-apposed plasma membrane (17), likely under the aegis of The histone deacetylase HDAC6 GTPases such as Rabs 7, 9 and 3 (18-22). depolymerizes tubulin, thereby destabilizing While unproven, studies in chicken microtubules. Cbl proteins compete with the osteoclasts raise the possibility that ruffled deacetylase for tubulin binding and thus border-forming vesicles may polarize to the promote polymerization (12). While resorptive surface via microtubules (23). The destabilizing microtubules in other cells (13), polarized vesicles fuse with the bone- RhoA appears to activate HDAC6 in glass- apposed plasma membrane, to increase its generated osteoclasts, suggesting the complexity, via a process mirroring GTPase negatively regulates the cell. When exocytosis (17). Similar to that occurring in on bone, however, osteoclasts in which the context of neurotransmitter exocytosis, RhoA is inhibited lose their apical-basal vesicle/plasmalemma fusion is regulated by polarity and, consequently, are incapable of v- (vesicular) and t- (target) SNAREs optimal resorption (6). Furthermore, RhoA (soluble N-ethylmaleimide-sensitive fusion promotes actin ring and podosome protein (NSF) attachment protein (SNAP) formation and osteoclast motility (6;14). receptors) (24). When osteoclasts contact bone, RhoA is activated and localizes to the cytoskeleton Ruffled border formation requires a (15;16), The fact that RhoA activation is synaptotagmin (Syt) linking the vesicle and diminished, in osteoclasts lacking αvβ3, target plasma membrane. Fifteen Syt establishes that the GTPase is regulated by isoforms have been identified in mammalian the integrin (3). Because of the conflicting cells but Syt VII generates ruffled borders phenotypes of osteoclasts on plastic or and is essential for bone degradation (17). bone, the impact of active RhoA on stability Syt VII also enables osteoblasts to secrete of their microtubules remains unknown. bone matrix proteins and as such, both resorption and formation are repressed in The Ruffled Border Is King Syt VII-deficient mice.

The ruffled border is the morphological sine Autophagy is a cellular degradative process qua non of the resorbing osteoclast as only by which cells recycle organelles and long- its presence assures that the cell is lived proteins. Autophagosomes, which are

IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 double-membrane bound vesicles, envelop β3(-/-) mice are hypocalcemic and and then deliver cellular components to osteoclast number is substantially increased lysosomes for degradation. While the in these animals, likely reflecting secondary process promotes survival in starved or hyperparathyroidism and an abundance of stressed cells as well as maintenance of osteoclastogenic cytokines in the marrow organelle quality (25-27), recent evidence (35;37;38). However, in contrast, absence of indicates it may also participate in regulated αv/β3 diminishes the abundance of the exocytosis (28). In fact, Atg5, Atg7 and polykaryons in vitro (33). As differentiation, LC3β autophagy proteins, representing the apoptosis and precursor proliferation are not two ubiquitin-like conjugation systems, are compromised, a reasonable hypothesis important for generation of the osteoclast holds that the paucity of osteoclasts, in ruffled border and the secretory function of culture, reflects cytoskeletal dysfunction, osteoclasts both in vitro and in vivo. Thus, and specifically impairment of migration osteoclasts lacking these proteins do not necessary for cell fusion. efficiently polarize cathepsin K to the resorptive microenvironment and are αv/β3 signaling in osteoclasts is initiated by incapable of optimal bone resorption (29). changing the integrin’s conformation from a low to a high affinity state by outside-in or Integrin Activity Starts It All inside-out activation (3;39). Outside-in activation is characterized by integrin Skeletal resorption requires osteoclast-bone clustering, thereby increasing avidity and recognition that is mediated by α/β affinity. Inside-out activation is an indirect heterodimers known as integrins. β1- event wherein signals emanating from containing heterodimers probably participate liganded growth factor or cytokine receptors in the process but αv/β3 is the key integrin target the integrin’s intracellular region, regulating skeletal degradation (30). While changing its conformation and consequently, the αv subunit is constitutively expressed that of the extracellular domain (40). As will throughout osteoclastogenesis, the be discussed, the adaptor protein, talin, is associated β chains alter with differentiation. essential for inside-out αv/β3 activation in Specifically, immature osteoclast precursors, osteoclasts and its absence arrests bone in the form of bone marrow macrophages, resorption. express abundant αvβ5 and little αvβ3. As the cells commit to the osteoclast Resorption is a cyclical event wherein a phenotype, the magnitude of expression of portion of the osteoclast migrates to a the two heterodimers reverses (31). Hence, candidate bone resorptive site and forms an αv/β3 is a relatively specific marker of actin ring and ruffled border. Following osteoclastogenesis. This integrin is liganded matrix degradation, the cell detaches and re- by the amino acids Arg-Gly-Asp (RGD), a initiates the cycle. Prior to bone recognition, motif present in bone proteins such as the integrin is predominantly in a low affinity osteopontin and bone sialoprotein. Small state and confined to podosomes within the molecules mimicking this sequence sealing zone (3;41). Activated αv/β3 leaves suppress osteoclast activity and are the podosome and transits to lamellipodia candidate anti-resorptive drugs (32-34). that mediate motility, compromised in the absence of the integrin (3). During In keeping with its governance of osteoclast resorption, the heterodimer appears in the function, absence of αv/β3, globally and ruffled membrane (3;41). conditionally in osteoclasts, increases bone mass and protects against estrogen- Integrins serve as attachment molecules but deficient osteoporosis (35;36). Reflecting the their intracellular transmission of matrix- integrin’s role in cytoskeletal organization, derived signals is at least as important. For αv/β3 deficiency yields deranged ruffled example, αv/β3 substrate robustly activates borders, failure of cell spreading and sub- ERKs in wild-type osteoclastic cells but not optimal bone resorption (35). Consequently, those lacking the integrin (3). Since

IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 activation of these MAP kinases typically regulated by the ubiquitinating activity of c- stimulates proliferation, their absence in β3 Cbl (50;51). knockout osteoclasts may contribute to their reduced numbers in vitro. The ITAM-bearing adaptors, Dap 12 and FcRγ, are expressed by osteoclasts and How Does αv/β3 Do It? their combined, but not individual deletion prompts severe osteopetrosis (45;52-54). In 1991, Soriano et al. determined that c-src While deletion of both co-stimulatory deletion eventuates in severe osteopetrosis molecules is reported to arrest due to osteoclast dysfunction (42). osteoclastogenesis (55), we find such is not Interestingly, c-src-deficient mice, like those the case (56), suggesting their resorptive lacking αv/β3, have increased numbers of abnormality reflects deranged osteoclast osteoclasts that fail to organize their function but not generation. The same cytoskeleton. c-src is a tyrosine kinase and obtains regarding osteoblast-mediated an adaptor protein and both functions are generation of osteoclasts lacking Dap12, necessary for optimal cytoskeletal with or without FcRγ. These mutant cells organization (43). form in normal numbers but fail to organize their cytoskeleton or resorb bone. Among Because c-src- and αv/β3-deficient the most dramatic consequences of this osteoclasts share qualitatively similar dysfunction is the inability of Dap12(-/-) cytoskeletal features, the kinase presents as osteoclasts to migrate through a layer of a mediator of integrin signaling. In fact, osteoblasts, required to attach to a under the aegis of phospholipase Cγ candidate resorptive bone surface (56;57). (PLCγ2) (44), c-src binds directly to the β3 Thus the dominant role of ITAM proteins in subunit in the bone resorptive cell, and we the osteoclast appears to be cytoskeletal have found this to be a constitutive event organization and not differentiation (56;58). (45). Others, however, propose that αv/β3 occupancy phosphorylates the focal Syk-mediated organization of the osteoclast adhesion kinase family member, Pyk2, cytoskeleton involves Vav3. This guanine which recruits c-src to the integrin (44;46). nucleotide exchange factor (GEF) is uniquely expressed in abundance in the cell αv/β3-associated c-src phosphorylates c-Cbl and activated upon v 3 occupancy in a which, in turn, inhibits c-src’s activity (3;47). α /β Regardless of the mechanism of SLP-76-dependent manner (15;59). Vavs association, c-src activation requires integrin transit cytoskeleton-organizing Rho GTPases from their inactive GDP- to their occupancy and again, signaling via PLCγ2. active GTP-associated conformation. Thus, Activated c-src prompts podosomal Vav3(-/-) osteoclasts are dysfunctional and disassembly, most probably by the mice from which they are derived are phosphorylating cortactin (48;49). Hence, osteopetrotic. podosomes are more abundant in c-src(-/-) than wild-type osteoclasts and the mutant Vavs are Rac GEFs and it is therefore not cells are less motile. In keeping with Pyk2 surprising that this Rho GTPase regulates regulating the cell’s cytoskeleton, osteoclasts lacking the kinase are unable to the osteoclast cytoskeleton in an αv/β3- generate normal sealing zones on bone dependent manner (60;61). The two (11). The cytoskeletal effects of Pyk2, isoforms expressed in osteoclasts, Rac1 however, may reflect its promotion of tubulin and Rac2, are mutually compensatory (62). acetylation. Effective deletion of both, however, produces severe osteopetrosis in which Syk is another non-receptor tyrosine kinase osteoclasts fail to organize their cytoskeleton. Absence of the related Rho mediating αv/β3 signaling in osteoclasts. family GTPase, cdc42, also causes Upon integrin occupancy it binds the β3 osteopetrosis but in this circumstance the cytoplasmic domain close to c-src, which dominant mechanism is arrested osteoclast activates it (45). Syk is also negatively

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Fig. 1. Proposed mechanism organizing the cytoskeleton of resorbing osteoclasts. 1). M-CSF occupying its receptor, c-fms, stimulates inside-out αvβ3 activation by inducing talin association with the β3 cytoplasmic domain that binds c-src constitutively. 2). Clustering of the integrin by RGD ligand increases avidity as well as affinity by outside-in activation. The liganded integrin activates c-src as evidenced by Y416 phosphorylation. Activated c-src tyrosine phosphorylates ITAM proteins that recruit Syk to the integrin by binding Syk-SH2 domains. c-src activates β3-associated Syk that phosphorylates Vav3 in the context of SLP-76. Vav3 then shuttles Rac-GDP to its activated GTP-associated state. 3). Rac-GTP prompts association of lysosome-derived secretory vesicles with microtubules (MTs) that deliver them to the bone- apposed plasma membrane into which they insert under the influence of Syt VII and LC3. Rac-GTP and MTs also promote sealing zone (SZ) formation. Secretory vesicle fusion focally expands the plasma membrane forming the ruffled border and eventuating in discharge of cathepsin K (CTK) and HCl into the resorptive microenvironment. recruitment due to inhibited precursor which M-CSF structures the osteoclast proliferation and accelerated apoptosis of cytoskeleton may, therefore, be independent the mature polykaryon (63). of the integrin, or alternatively, represent inside-out αv/β3 activation. In fact, M-CSF M-CSF Helps αv/β3 transits αv/β3 from its default low affinity to its high affinity conformation by inducing RANK ligand (RANKL) and M-CSF are the talin binding to the β3 cytoplasmic domain requisite osteoclastogenic cytokines but (39). Absence of talin, in osteoclast each also promotes the resorptive activity of precursors, does not arrest differentiation the mature polykaryon. In the case of M- but blocks substrate adherence and motility. CSF, the cytokine interacting with its The impaired function of talin-deficient receptor, c-fms, stimulates a signaling osteoclasts results in a 5-fold increase in the pathway remarkably similar to that induced bone mass of mutant mice. Interestingly, the by αv/β3, thereby organizing the osteopetrotic phenotype of mice with talin cytoskeleton (37;58;64). The means by (-/-) osteoclasts is more severe than of those

IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 lacking αv/β3, which likely represents arrest 3. Faccio R, Novack DV, Zallone A, Ross of compensatory integrins, particularly those FP, Teitelbaum SL. Dynamic changes in bearing β1 (30;35). the osteoclast cytoskeleton in response to growth factors and cell attachment Conclusion are controlled by beta3 integrin. J Cell Biol. 2003 Aug 4;162(3):499-509. The magnitude of bone resorption reflects osteoclast number and function of the 4. Saltel F, Chabadel A, Bonnelye E, individual cell, the latter dependent upon Jurdic P. Actin cytoskeletal organisation cytoskeletal organization. The osteoclast in osteoclasts: a model to decipher cytoskeleton is a unique structure whose transmigration and matrix degradation. conversion to its active state depends upon Eur J Cell Biol. 2008 Sep;87(8-9):459- contact with mineralized matrix (Fig. 1). 68. These extracellular signals, which polarize the resorptive machinery to the bone-cell 5. Chabadel A, Bañon-Rodríguez I, Cluet interface, are transmitted intracellularly by D, Rudkin BB, Wehrle-Haller B, Genot integrins dominated by αv/β3. In conjunction E, Jurdic P, Anton IM, Saltel F. CD44 with M-CSF-stimulated inside-out activation, and beta3 integrin organize two a canonical signaling pathway emanates functionally distinct actin-based domains from the αv/β3 integrin. Occupancy of the in osteoclasts. Mol Biol Cell. 2007 heterodimer phosphorylates constitutively Dec;18(12):4899-910. associated c-src which in turn targets Dap 12. The ITAM’s phosphotyrosines serve to 6. Saltel F, Destaing O, Bard F, Eichert D, recruit Syk to the β3 cytoplasmic domain Jurdic P. Apatite-mediated actin where it is also phosphorylated by c-src. dynamics in resorbing osteoclasts. Mol Utilizing SLP-76, Syk activates Vav3, Biol Cell. 2004 Dec;15(12):5231-41. eventuating in formation of Rac-GTP and organization of the resorptive cytoskeleton. 7. Destaing O, Sanjay A, Itzstein C, Horne Given current concerns regarding long- WC, Toomre D, De Camilli P, Baron R. acting anti-resorptive agents, such as The tyrosine kinase activity of c-Src bisphosphonates, short-acting counterparts regulates actin dynamics and are in demand. Delineating the molecular organization of podosomes in mechanism by which osteoclasts organize osteoclasts. Mol Biol Cell. 2008 their cytoskeleton to degrade bone has Jan;19(1):394-404. provided an array of candidate therapeutic targets. 8. Luxenburg C, Geblinger D, Klein E, Anderson K, Hanein D, Geiger B, Conflict of Interest: None reported. Addadi L. The architecture of the adhesive apparatus of cultured Peer Review: This article has been peer-reviewed. osteoclasts: from podosome formation to sealing zone assembly. PLoS ONE. References 2007 Jan 31;2(1):e179.

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REVIEW

Osteoclast Activity and Subtypes as a Function of Physiology and Pathology—Implications for Future Treatments of Osteoporosis

K. Henriksen, J. Bollerslev, V. Everts, and M. A. Karsdal

Nordic Bioscience A/S (K.H., M.A.K.), DK-2730 Herlev, Denmark; Section of Endocrinology (J.B.), Department of Medicine, Rikshospitalet, Oslo University Hospital and the University of Oslo, 0450 Oslo, Norway; and Department Oral Cell Biology (V.E.), Academic Centre of Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, Research Institute Move, 1066 EA Amsterdam, The Netherlands

Osteoclasts have traditionally been associated exclusively with catabolic functions that are a prerequisite for bone resorption. However, emerging data suggest that osteoclasts also carry out functions that are important for optimal bone formation and bone quality. Moreover, recent findings indicate that osteoclasts have different subtypes depending on their location, genotype, and possibly in response to drug intervention.

The aim of the current review is to describe the subtypes of osteoclasts in four different settings: 1) physiological, in relation to turnover of different bone types; 2) pathological, as exemplified by monogenomic disorders; 3) pathological, as identified by different disorders; and 4) in drug-induced situations.

The profiles of these subtypes strongly suggest that these osteoclasts belong to a heterogeneous cell population, namely, a diverse macrophage-associated cell type with bone catabolic and anabolic functions that are dependent on both local and systemic parameters. Further insight into these osteoclast subtypes may be important for understanding cell–cell communication in the bone microenvironment, treatment effects, and ultimately bone quality. (Endocrine Reviews 32: 31–63, 2011)

I. Introduction F. Other diseases characterized by increased oste- II. Bone Remodeling oclast activity III. The Classical Osteoclast VI. Drug-Induced Osteoclast Subtypes A. Existing drugs IV. Osteoclast Subtypes in Physiological Situations B. Future treatments A. Endochondral vs. intramembranous bone VII. The Bone Anabolic Effects of the Osteoclasts osteoclasts VIII. Conclusions and Future Perspectives B. Chondroclasts C. Osteoclasts involved in targeted and stochastic remodeling D. Trabecular and cortical osteoclasts I. Introduction E. Diurnal variation in osteoclasts or osteoclast activity? steoclasts are multinucleated bone-resorbing cells V. Osteoclast Subtypes in Pathological Situations O that are unique in their ability to degrade mineral- A. Osteoporotic osteoclasts ized matrices, such as bone and calcified cartilage (1). Os- B. Changes in osteoclast activities with increasing teoclasts have for a long time been considered bone- bone matrix age C. Osteoclast-rich osteopetrosis resorbing “machines,” yet some years ago it was D. Osteoclast-poor osteopetrosis demonstrated that not all osteoclasts are the same and that E. Pycnodysostotic osteoclasts careful elucidation of the osteoclast subtype may prove

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: AGE, Advanced glycation end-product; BMD, bone mineral density; CAII, car- Printed in U.S.A. bonic anhydrase II; ClC-7, chloride channel 7; CT-1, cardiotrophin-1; CTX, C-terminal Copyright © 2011 by The Endocrine Society crosslinked telopeptide of type I collagen; ER, estrogen receptor; GLP, glucagon-like peptide; doi: 10.1210/er.2010-0006 Received March 19, 2010. Accepted August 10, 2010. HRT, hormone replacement therapy; ICTP, carboxyterminal peptide of type I collagen; MMP, First Published Online September 17, 2010 matrix metalloproteinase; ONJ, osteonecrosis of the jaw; OPG, osteoprotegerin; RA, rheumatoid arthritis; RANK, receptor activator of nuclear factor ␬B; RANKL, RANK ligand; SERM, selective estrogen receptor modulator; TRACP, tartrate-resistant acid phosphatase; V-ATPase, vacuolar type ATPase.

Endocrine Reviews, February 2011, 32(1):31–63 edrv.endojournals.org 31 32 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31–63 beneficial (1–4). As illustrated in Table 1, under normal that cannot be completely countered by an increase in circumstances, osteoclasts are influenced by a complex bone formation. This results in low bone mass, in deteri- combination of systemic hormones and local mediators oration of the microarchitecture of the skeleton, and often present in the different bones. Under pathological condi- in fractures (25). Osteoporotic fractures are associated tions, such as cessation of estrogen production or inflam- with increased morbidity and mortality and give rise to a matory conditions, additional cytokines are present. Even significant public health problem (24). the bone type and age may influence the phenotype of the Osteopetrosis, on the other hand, is a rare, inherited osteoclast (2, 5–7). Finally and importantly, different disease in various species including man, which was orig- classes of drugs used for treatment of osteoporosis and inally identified by Albers-Scho¨ nberg in 1904 (26). In the other diseases also influence the osteoclasts significantly. majority of cases, it is caused by defective resorption by the The aim of this review is to provide a thorough descrip- osteoclasts, resulting in high bone mass with poor bone tion of the complex nature of osteoclasts under healthy quality and increased fracture frequency due to defective and diseased states and to describe their modulation by bone remodeling (1, 26, 27). However, recent studies also drugs that have been approved for use or are under de- characterized patients with osteopetrosis due to dys- velopment. The paper will also emphasize the role of os- functional osteoclastogenesis either directly affecting teoclasts in initiating bone formation, a recently discov- the osteoclast precursors or indirectly through the os- ered activity of these cells that has gained much attention teoblasts, and thus the phenotype was caused by the (3, 8–13). absence of osteoclasts, rather than inactivity of these cells (28–31). Interestingly, the studies of osteopetrotic patients have indicated that the presence of osteoclasts, II. Bone Remodeling but not their activity, is essential for bone formation, indicating that some aspects of the coupling principle Bone remodeling is required for optimal control of cal- should be revised (1, 3). cium homeostasis and strength of the bones and is essential Because hypogonadal osteoporosis is associated with for the continued maintenance of a healthy skeleton (14). increased numbers and activity of osteoclasts (16), most Bone remodeling is performed by three cell types: 1) the treatments developed so far, such as bisphosphonates and osteoclasts, which are the sole cells in the body possessing hormone replacement therapy (HRT)/selective estrogen the ability to degrade both the inorganic calcium matrix receptor modulators (SERMs), have focused on eliminat- and the organic collagen matrix; 2) the osteoblasts, which ing or reducing the number of osteoclasts and thereby are the bone-forming cells; and 3) the osteocytes, which reducing bone resorption (32). These treatments are as- appear to regulate the activity of both osteoclasts and os- sociated with secondary decreases in bone formation due teoblasts (14, 15). In healthy adults, under normal cir- to the coupled nature of the bone remodeling process, cumstances, bone resorption is always followed by an which naturally limits their efficacy (3, 24). However, as equal degree of bone formation, a tightly balanced process seen in the osteopetrotic syndromes, there are indications referred to as coupling (9, 16). The modulation of activ- that bone resorption and bone formation can be dissoci- ities of the cells involved in the remodeling cycle was re- ated, and from recent studies it appears that the osteoclast cently described in detail (15). itself, whether it is a physiological, pathological, or drug- Coupling was initially discovered in the 1960s when induced subtype, is highly important for a secondary effect Frost and co-workers (17, 18) demonstrated that osteo- on bone formation (1, 3). blasts filled the resorption pits created by osteoclasts in In this review, we describe differences in osteoclast more than 97% of the cases (17–21). Since then, coupling activity and subtypes in relation to physiology, patho- has been understood as a coordinated and balanced in- physiology, and medication, with special attention to duction of osteoblastic bone formation in response to coupling in the bone remodeling process. Ultimately, prior bone resorption (19). Uncoupling occurs when the this review highlights potential directions for new treat- balance between resorption and formation is disrupted, ment modalities. which often leads to pathological situations such as osteoporosis or osteopetrosis (3, 9, 14, 22, 23). However, uncoupling also occurs under physiological conditions, III. The Classical Osteoclast i.e., during skeletal growth in children, where bone formation exceeds bone resorption (10). Osteoclastogenesis is a complex process requiring both the Hypogonadal osteoporosis is usually caused by a de- correct extracellular stimuli and the correct cellular mol- crease or loss of sex steroid production, which results in ecules to interact without impediment (22, 33). Osteoclasts accelerated osteoclastogenesis and bone resorption (24) arise from hematopoietic stem cells that, in the presence of Endocrine Reviews, February 2011, 32(1):31–63 edrv.endojournals.org 33

TABLE 1. A simplified summary of osteoclast phenotypes as a function of physiology, pathology, and drugs, also indicating areas of osteoclast biology that are not well-understood

Osteoclast no. Bone formation Resorptive process Bone resorption Acid Cat K MMP Classical osteoclast Normal Normal ϩϩϩ ϩϩϩ ϩ/Ϫ Balanced Physiology Targeted Recruitment to specific Increased ϩϩ ? ? Balanced areas increased Stochastic Not clear Not clear ϩ ? ? Not clear Night Normal Increased ϩϩ ϩϩ ϩ/Ϫ Minor up-regulation Day Normal Decreased ϩ ? ? Minor down-regulation Chondroclast Normal Normal ϩϩϩϩBalanced Endochondral Normal Normal ϩϩ ϪBalanced Intramembraneous Normal Normal ϩϪ ϩNot balanced, opposite side of bone than resorption Trabecular Normal Normal ϩϩ ϩϩ Ϫ Balanced Cortical Normal Normal ϩϩ ϩϩ Ϫ Balanced Pathology Osteoporosis Increased Increased ϩϩϩϩ Ϫ Increased, but less than resorption Age Increased Increased ϩϩ ϩϩ ? Decreased OC-rich OP Greatly increased Decreased ϪϪ ϪIncreased according to increased OC number OC-poor OP No osteoclasts Decreased ϪϪ ϪDecreased? Pycnodysostosis Unchanged/increased Decreased ϩ Ϫ ϩϩϩ Not clear osteoclast size Paget’s Greatly increased at Increased at local sites ϩϩ ϩ ϩϩϩ Increased locally, but local sites does not compensate resorption RA Greatly increased at Not known ϩ ϩ ϩϩϩ Not known local sites Lytic metastases Greatly increased at Increased at local sites ϩ ϩ ϩϩϩ Increased locally, but local sites does not compensate resorption Drug-induced BPs Reduced Decreased ϪϪ ? Decreased secondary to resorption HRT/SERMs Reduced Decreased ϪϪ ? Decreased secondary to resorption Calcitonin Unchanged Decreased ϪϪ ? Not changed or minor decrease PTH Increased/unchanged Decreased ϩϩ ϩϩ ? Increased, but only temporarily Strontium ranelate Unchanged Decreased? ϪϪ ? Increased GCs Unchanged/increased Unchanged/increased ϩϩ ? Decreased strongly Denosumab Greatly reduced Decreased ϪϪ ϪDecreased secondary to resorption Cat K inhibitors Unchanged Decreased ϩ Ϫ ϩϩϩ Decreased secondary to resorption GLP-2 Unchanged Decreased ϪϪ ? Not changed, but long term effects are not known Acidification inhibitors Increased Decreased ϪϪ ϪIncreased, but so far only in animal models

The table shows the subtype of osteoclasts, the number of osteoclasts, the effect on bone resorption, which part of the resorption machinery that is active/affected, and the effect on bone formation. OC, Osteoclast; OP, osteopetrosis; Cat K, cathepsin K; BPs, bisphosphonates; GCs, glucocorticoids; MMP, matrix metalloproteinase; RA, rheumatoid arthritis; PTH, parathyroid hormone; HRT, hormone replacement therapy; SERMs, selective estrogen receptor modulators; GLP-2, glucagon-like peptide 2. 34 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31–63

FIG. 1. Schematic illustration of the molecules involved in osteoclastogenesis and function. (See Refs. 1, 33, and 42). NF␬B, nuclear factor ␬ B; TRAF6, TNF receptor-associated factor 6; nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1; PYK2, Proline-rich Tyrosine Kinase 2; MitF, microphthalmia-associated transcription factor; ClC-7; Chloride Channel 7; PLEKHM1, pleckstrin homology domain containing, family M (with RUN domain) member 1; osteopetrosis associated transmembrane protein 1; CA2, carbonic anhydrase II; AE2, anion exchanger 2. receptor activator of nuclear factor ␬B (RANK) ligand teolysis, although these processes likely occur at the same (RANKL) and macrophage-colony stimulating factor, un- time (44, 51). dergo differentiation and fusion resulting in large multinu- Bone resorption is initiated by active secretion of pro- cleated cells characterized by expression of a series of oste- tons through a vacuolar type ATPase (V-ATPase) and pas- oclast markers, such as tartrate-resistant acid phosphatase sive transport of chloride through a chloride channel (52, (TRACP), matrix metalloproteinase (MMP)-9, cathepsin K, 53). The secretion of hydrochloric acid leads to dissolution carbonic anhydrase II (CAII), the a3 subunit of the vacuolar of the inorganic matrix of the bones (54). The osteoclastic [Hϩ]-ATPase, chloride channel 7 (ClC-7), osteopetrosis-as- V-ATPase is functionally specific and contains the a3 sub- sociated transmembrane protein 1, and the calcitonin recep- unit, and accordingly, loss of a3 leads to osteopetrosis (37, tor (34–41). Osteoclastogenesis and the molecules involved 55–57). In both mice and man, the chloride channel ClC-7, in this process are summarized in Fig. 1, but are not has been shown to mediate chloride transport, thereby discussed in any further detail because several excellent ensuring the electrochemical balance required for intense reviews have been published recently on this topic (1, acidification (Fig. 2) (8, 39, 58). Recent data showed that 22, 33, 42). ClC-7 functions as a proton-chloride antiporter (59, 60). Polarization and formation of the sealing zone, which To generate the necessary levels of Hϩ and ClϪ, the is a specialized ring structure containing a high number of enzyme CAII catalyzes conversion of CO2 and H2O into ␤ ϩ Ϫ -actin filaments, are the next steps in the life span of an H2CO3, which ionizes into H and HCO3 (35), thereby ␣ ␤ osteoclast (43, 44). These processes require the v 3 in- providing the protons for the V-ATPase (27). Meanwhile, Ϫ Ϫ tegrin and the intracellular signal transducers c-src, Syk, basolateral exchange of HCO3 ions for Cl by anion ex- and proline-rich tyrosine kinase 2, as well as the microph- changer 2 (61–63) provides ClϪ ions required for the in- thalmia transcription factor (33, 45, 46), which appears to tense acidification occurring in the resorption lacuna. be an important regulator of osteoclastic gene transcrip- Interestingly, long bones differ from flat bones with tion (47–49). respect to the molecular nature of the acidification ma- The final step of osteoclastogenesis is the activation of chinery (62). resorption, a process that is characterized by the forma- Proteolysis of the type I collagen matrix in bones is tion of a ruffled border that is an intensely convoluted mainly mediated by the cysteine proteinase, cathepsin K. membrane present inside the sealing zone (43, 44). The This enzyme is active at low pH in the resorption lacuna formation of the ruffled border is not well-characterized; (Fig. 2) (64–68). The neutral MMPs also appear to play a however, the signaling molecule Rab7 is required (50). minor role during organic matrix degradation; however, Bone resorption takes place at the ruffled border local- the exact role of MMPs is still being investigated (69) and ized at the apical side of the osteoclasts, and it can be is highly dependent on the bone type (38, 70, 71). The divided into two processes, namely acid secretion and pro- resorbed material is removed from the resorption pit by Endocrine Reviews, February 2011, 32(1):31–63 edrv.endojournals.org 35

FIG. 2. Top, Schematic illustration of the differences between acid secretion and proteolysis during osteoclastic bone resorption, illustrating that the collagen matrix is removed by proteolysis after acidification. Bottom, Mutations/knockout in genes/proteins involved in bone resorption, phenotypes, and effect on osteoclasts. Ae2, Anion exchanger 2; a3 V-ATPase, a3 subunit of the osteoclast-specific V-ATPase. ClC-7, chloride channel 7; OSTM1, osteopetrosis associated transmembrane protein 1; CA2, carbonic anhydrase 2; MMP-9, matrix metalloproteinase 9. uptake and transcytosis through the osteoclast (72, 73). IV. Osteoclast Subtypes in After completing resorption, osteoclasts either undergo Physiological Situations apoptosis or perform a further round of resorption (44) Osteoclast activities are essential for development, as well (Table 1). as remodeling of bone in response to aging and stress (6, In summary, the osteoclasts are highly specialized for 14, 15). Under normal physiological circumstances, the both dissolution of the inorganic matrix and degradation osteoclasts can be categorized into subgroups depending of the organic matrix of the bones. These highly polarized on the matrix on which they are positioned, the time of cells are characterized by a unique set of membrane-bound day, and the type of remodeling in which they participate. molecules that ensure an efficient resorption of bone and These different groups of osteoclasts have provided key other mineralized tissues. This complex machinery may be information on skeletal maintenance. affected by a range of important parameters in physiology and pathology, and importantly in drug-induced situa- A. Endochondral vs. intramembranous bone osteoclasts tions that are important to identify and advance osteoclast Anatomically, two types of bones are present in the research and biology. body, the long bones (e.g., the femur and tibia) and the 36 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31–63 flat bones (e.g., the calvarium), with the main difference chondroclasts may also play a role in both rheumatoid between these two types of bone being their develop- arthritis (RA) and osteoarthritis (83–85). The term chon- ment (74). Studies also indicate that osteoclasts on these droclast derives from the localization of these cells on cal- two types of bones are functionally different with re- cified cartilage as seen in the expanding growth plates spect to both acid secretion and proteases involved in during endochondral ossification (80, 86). For their for- degradation (2). mation, these cells are dependent on the presence of Evidence that differences between resorption of flat and macrophage-colony stimulating factor and RANKL, as long bone exist was presented in 1999 (4). However, in- are bone-resorbing osteoclasts (87–90). dications that even the acidification process is different Most of the evidence for the functionality of chondro- have been published only recently (62). Data from mice clasts is derived from studies of the longitudinal growth of deficient in the bicarbonate-anion exchanger Ae2 (Slc4a2) long bones, i.e., metatarsals and tibias isolated from have shown that it is essential for bone resorption in long mouse embryos (38, 80, 91–93). First, bone/cartilage re- bones (61, 63), whereas it is not involved in bone resorp- sorption in these models is still dependent on acid secretion tion in calvariae (62), showing that distinct acid transport as evidenced by mice with mutations in the acid secretion mechanisms are present in different subsets of osteoclasts. process, i.e., ClC-7-deficient and a3 V-ATPase-deficient With respect to acid secretion into the resorption lacunae, mice, as well as mice unable to form sealing zones, i.e., it is presently not known whether any differences exist, c-src-deficient mice, in which the massive bones mainly although the absence of calvarial thickening in patients consist of calcified cartilage due to the defective resorption with defective ClC-7 strongly suggests that ClC-7 is not process (56, 94, 95). Similar findings have been noted in involved in resorption of the flat bones (75). the corresponding human disease(s) (27, 96, 97). Interest- Extensive research into the proteolytic processes involved in resorption of flat and long bones clearly dem- ingly, the ruffled border is less prominent in the chondro- onstrates that the proteolytic processes involved in deg- clasts than osteoclasts, potentially suggesting that lower radation of these two types of bone matrix are distinct (4, levels of acid secretion are required for dissolution of this 71). Osteoclasts in flat bones preferentially appear to en- matrix (98, 99). The main difference between chondro- gage in MMP-mediated bone resorption, although cathep- clasts and osteoclasts is in the profiles of enzymes neces- sin L seems to be involved, too. Osteoclasts in long bones sary for tissue degradation. Resorption by chondroclasts primarily depend on cysteine proteinases, in particular ca- does not appear to depend as much on cathepsin K as does thepsin K (4, 71). TRACP also appears to be involved in resorption by osteoclasts. Cathepsin K-deficient mice bone resorption, and more so in calvarial bone (76–78). show no evidence of calcified cartilage in the marrow cav- When osteoclasts generated from human peripheral blood ity of long bones, indicating that the removal of calcified are seeded on cortical bone, they primarily depend on ca- cartilage during endochondral ossification occurs, al- thepsin K, whereas when cathepsin K activity is blocked, though there are indications that the process is delayed there is some compensatory bone resorption mediated by (65, 100). Of importance is the observation of a massive MMPs (69). How these osteoclasts behave on bone sub- compensation by MMPs in the absence of cathepsin K, strates other than cortical bone is presently not known which obscures the interpretation of data from cathepsin (Table 1). K-deficient systems (69, 70, 101–103). Finally, one study Data suggesting that the bone matrix could play a role has indicated that TRACP is activated and secreted at the in the control of osteoclastic activities were presented in a ruffled border in cathepsin K-deficient osteoclasts, and study showing compositional differences between long only in cells that play a role in the removal of calcified bone and flat bone matrices, including differences in the cartilage (104) (Table 1). presence of putative cysteine proteinase inhibitors (79). Although chondroclasts are involved in the degrada- The functional significance of these data still remains to be tion of a different matrix than osteoclasts, an interesting fully elucidated, although they clearly illustrate the im- observation is that bone formation is tightly coupled to portance of understanding how a given context affects the resorption of the mineralized matrix, as has clearly been osteoclasts. demonstrated in studies of endochondral ossification (86). B. Chondroclasts It is more likely that bone formation is coupled to chon- It has long been discussed whether chondroclasts are a droclast numbers because release of molecules from deg- “real” cell type or whether they simply are osteoclasts that radation of cartilage, which in composition is far from reside on cartilage instead of bone (77, 80–82). Chon- bone, would be expected to be different from molecules droclasts are mainly important in endochondral bone de- released during bone resorption; however, this has never velopment, and in addition there is some evidence that been studied in detail. Endocrine Reviews, February 2011, 32(1):31–63 edrv.endojournals.org 37

FIG. 3. Schematic illustration of stochastic vs. targeted remodeling. The figure illustrates the local nature of targeted remodeling, which is activated at specific sites after the formation of microcracks and leads to removal of the microcrack and restoration of the damaged bone. Stochastic remodeling, on the other hand, is of a systemic nature and is activated by low calcium levels in the circulation leading to PTH release. Other hormones, such as vitamin D3 (VitD3), and potentially calcitonin also play roles in stochastic remodeling. It appears that there are two levels of calcium homeostasis—one mediated by osteocytes independent of osteoclasts, and one including the osteoclasts—although the balance between these two ways of releasing calcium still remains to be fully understood.

C. Osteoclasts involved in targeted and ized by high numbers of apoptotic osteocytes, are prefer- stochastic remodeling entially and rapidly degraded by osteoclasts. This further Two different modes of remodeling have been pro- supports the possibility that changes in the bone matrix posed: targeted and stochastic. Targeted bone remodeling and the balance between live and dead osteocytes deter- takes place at specific sites, whereas stochastic remodeling mine which areas will be remodeled (108). Finally, a recent occurs more randomly (19, 105, 106). The first type is study demonstrated that targeted ablation of osteocytes primarily performed to replace microdamaged bone and led to a dramatic increase in osteoclast activity (109) (Ta- thus to maintain the load-bearing capacity of the skeleton. ble 1). These data indicate that death of osteocytes is a key The second type of remodeling appears random with re- point in induction of osteoclast activity (Fig. 3). spect to localization, although it may be involved in main- Taken together with the finding that bones of different taining integrity of the bones, independent of damage. age lead to different levels of osteoclastogenesis (7), it ap- This process is hormonally regulated (105, 107). pears that osteoclast functionality is at least partially con- The balance between these two modes of bone remod- trolled by osteocyte-derived molecules, which are seques- eling has not been fully elucidated yet, but studies in dogs indicate that approximately 30% of all remodeling is tar- tered in the bone matrix. geted and the remaining 70% is stochastic (105). With Stochastic remodeling, while occurring at random sites, respect to the osteoclasts mediating these two types of is centrally regulated by hormones such as PTH, vitamin remodeling, most studies have focused on how targeted D3, and potentially calcitonin, and its main role is the remodeling is controlled. regulation of calcium homeostasis (110–112). It has even It appears that the bone matrix contains signals regu- been questioned to what extent this process depends on the lating the activity of the osteoclasts (108). A recent study presence of osteoclasts because patients with nonfunc- demonstrated that aged bones were more readily resorbed tional osteoclasts have normal calcium homeostasis (110– than young bones, thus supporting the hypothesis that the 112). Yet, when they are calcium-deprived, osteopetrotic bone matrix composition influences remodeling rates (7). patients fail to correct their calcium levels, indicating that Furthermore, areas of microdamage, which are character- osteoclasts, which are either absent or nonfunctional in 38 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31–63 these patients, do play a role in calcium homeostasis, and number and activity (32) (Table 1). An interesting aspect of thus stochastic remodeling (Fig. 3) (113). this is that targeting nocturnal resorptive activity appears to In summary, targeted remodeling is beginning to be lead to inhibition of bone resorption, whereas not attenuat- understood in detail, and it is a tightly regulated and cou- ing bone formation (125–127), thereby highlighting an in- pled process involving osteocytes, osteoclasts, and cells of teresting prospect of reducing bone resorption in a specific, the osteoblast lineage. On the other hand, stochastic re- nocturnal manner. modeling and the role it plays in calcium homeostasis are In summary, studies of osteoclasts under different phys- still not very well understood, although there are indica- iological conditions, such as those listed above, have high- tions that there is a level of regulation by the activity of lighted the heterogeneity of these cells. Furthermore, these osteoclasts. studies highlighted the importance of the balance between bone resorption and bone formation, a tightly regulated D. Trabecular and cortical osteoclasts phenomenon that rarely is disturbed under physiological Bone remodeling does not occur with the same fre- conditions. Finally, how the heterogeneity of the oste- quency in cortical and trabecular bone. Every year, 25% oclasts affects bone formation is presently not well under- of the trabecular bone matrix, but only 4% of the cortical stood, but a further understanding of this process could matrix, is remodeled (114). Interestingly, most in vitro help optimal treatment of diseases involving alterations in osteoclast experiments are based on cortical bone (or den- bone remodeling. tine) substrates, which are either slowly remodeled or not remodeled at all (7, 102, 114–116). Studies have shown that bones endogenously contain signals regulating osteo- V. Osteoclast Subtypes in clastogenesis and resorption and that these signals appear Pathological Situations to be related to the age of the bone (7, 117) (Table 1). Thus, an interesting question is whether osteoclasts themselves Changes in osteoclast activity and number have been de- are indeed different when derived from different matrices tected in several diseases, ranging from illnesses involv- or whether the difference is matrix related. Furthermore, ing excessive bone resorption, such as osteoporosis and systemic regulation is likely to be involved in controlling Paget’s disease; to those involving secondary activation of which bones are resorbed to some extent. osteoclasts, such as osteolytic metastases and RA; to dis- These data also correlate with evidence indicating that eases involving defective osteoclast differentiation and/or remodeling of different bone compartments can be either function, such as osteopetrosis. These different types of primarily targeted, such as in the cortex, or primarily sto- diseases have shed important light on osteoclastic function chastic, as seen in some parts of trabecular bone (106). A with respect to obtaining the right type of treatment. They further understanding of this could provide directions for have also shed light on a very central aspect in bone biol- the development of novel drugs producing optimal benefit ogy, the coupling principle. The coupling principle de- at the sites where it is most needed, i.e., leading to a better scribes the phenomenon that bone formation follows bone fracture reduction than that presently obtained. resorption, which leads to a complete restoration of the bone removed during bone resorption (17).

E. Diurnal variation in osteoclasts or osteoclast activity? Bone resorption markers measured in serum may be A. Osteoporotic osteoclasts interpreted as indicating the net result of all osteoclast sub- 1. Changes in osteoclastogenic potential in osteoporosis types and activity levels at one particular time. A wide range An important aspect of osteoporosis is whether the of factors, known and unknown, may influence the inter- number of osteoclast precursors in the circulation in- pretation (15). Diurnal variation is a well-established and creases, and, if so, whether the osteoclastogenic potential important parameter of bone turnover. Postprandially, bone of these cells is increased. Eghbali-Fatourechi et al. (128) resorption decreases by approximately 50% compared with showed that the overall number of cells expressing that of fasting individuals, but during the night, bone resorp- RANKL is increased in postmenopausal women com- tion increases to an equally large degree (118–120). Several pared with premenopausal or estrogen-treated women, investigations have demonstrated that the circadian varia- clearly indicating that the bone marrow microenviron- tion in bone resorption is induced in part by food intake ment, including stromal, T, and B cells, changes in a proos- (121–123), which, at least partially, involves the peptide hor- teoclastic direction when estrogen is reduced. These data mone glucagon-like peptide (GLP) 2 (124). Interestingly, the were supported by a recent study from the same authors osteoclast number does not appear to depend on the time of showing that bone marrow cells isolated from estrogen- day, further emphasizing differences between osteoclast treated or control postmenopausal women displayed Endocrine Reviews, February 2011, 32(1):31–63 edrv.endojournals.org 39 reduced osteoclastogenic potential (129) (Table 1). Es- of osteoclastogenesis (137). Again, there appear to be dif- trogen was shown to have a dual mode of action—the ferent osteoclastic subtypes, which also appear to be rel- first leading to overall lower RANKL expression by evant in the context of bone loss rates in different bone bone marrow cells, and the second reducing the oste- compartments during osteoporosis (114). As indicated oclastogenic response to RANKL (130). Interestingly, above, an important point is the difference between gen- aging of mice was also shown to increase the osteoclas- ders (139). In mice, the gender-based difference between togenic potential of bone marrow cells, both by up- cortical apposition and endocortical resorption that be- regulation of RANKL production and by increasing comes more apparent with increasing age might be ex- precursor sensitivity to RANKL (131). plained by differences in ER expression (142). With re- In vitro studies of the changes in cellular activity of spect to changes in the osteoclasts after menopause, a osteoclasts from osteoporosis patients are limited, but couple of studies have clearly demonstrated that bone re- these have indicated both an accelerated osteoclastogen- sorption, as well as bone formation, increases in women esis and resorption (132, 133). Furthermore, a key cell in after menopause (143–145), and these changes become the up-regulation of osteoclastogenesis appears to be the more explicit in high- and low-turnover patients (145). T cell, which responds to lowered estrogen levels by in- Although bone formation increases as a function of the creasing RANKL production (134). increased resorption, it does not match bone resorption, The main issue with all the studies of osteoporotic os- thereby illustrating the importance of understanding the teoclasts and their precursors is the use of mixed cell pop- interplay between osteoclasts and osteoblasts in detail. ulations, which clouds the interpretation of the results, and therefore these aspects of osteoclastic function still B. Changes in osteoclast activities with increasing bone require further investigation. Furthermore, with the recent matrix age publication of the possibility of assessing the “anabolic Numerous studies have investigated the control of os- potential” of osteoclasts (11, 13), it would be of interest to teoclast activity as a function of changes in biochemical investigate the anabolic capacity of osteoporotic oste- properties of the bone matrix. Aging leads to accumula- oclasts and thus shed light on the imbalance between bone tion of different biochemical modifications of the bone resorption and bone formation in osteoporosis. matrix, such as advanced glycation end-products (AGEs), 2. A direct role for sex steroids on osteoclasts homocysteine, increased calcium concentration, as well as The role of estrogen on cells belonging to the osteoclas- some modifications of the collagen matrix (146). tic lineage has been studied extensively, with several find- Recent studies have indicated that these modifications ings indicating that estrogen suppresses osteoclastogenesis of the bone matrix itself actually modulate the activity of but not the resorptive activity of mature osteoclasts (135– the osteoclasts to a certain extent (7, 117). Homocysteine, 137). Androgens, such as dihydrotestosterone, exhibit which accumulates in bone and in circulation with age, similar effects to estrogen on osteoclasts in vitro, although was shown to activate osteoclastogenesis and bone resorp- this has not been studied in great detail (138). Finally, a tion (147) (Table 1). AGEs are modifications of proteins recent study using mice deficient for the estrogen receptor that accumulate in various tissues with age, and they have (ER)-␣, specifically in mature osteoclasts, showed bone been implicated in the pathology of osteoporosis (146, loss in female, but not male, mice (139). This demon- 148). Some evidence indicating a direct regulation of os- strated that estrogen likely plays a direct role in bone re- teoclast activity by AGEs has been published, but these sorption by even mature osteoclasts (139). Although the studies are contradictory. One study shows activation of authors used the cathepsin K promoter to ensure specific resorption by AGE-modified proteins (149), whereas the knock-down of the ER-␣ in osteoclasts, cathepsin K is also other study shows the opposite (117); however, quite dif- expressed in preosteoclasts, albeit to a lower extent (139). ferent techniques were used. More studies are needed to investigate the role of ER-␣ in Interestingly, AGEs are accumulated in diabetes, and mature osteoclasts specifically. Interestingly, osteoclasts they have been speculated to be involved in the increased in different bone sites preferentially express different ERs, fracture rates observed in patients with this disease (150, with cortical osteoclasts mainly expressing ER-␣ and 151). Another intriguing finding is the induction of trabecular osteoclasts mainly expressing ER-␤ (140), apoptosis in osteoblasts by AGEs (152), which potentially whereas from a functional point of view ER-␣ appears to could play a role in the imbalance between osteoclast and be more relevant for trabecular, not cortical, bone (141). osteoblast function during osteoporosis and aging. These Furthermore, the expression pattern also differs between findings are all preliminary in nature, and they await con- mature and differentiating osteoclasts; ER-␣ is mainly ex- firmation from independent research groups. However, pressed in immature cells, and ER-␤ is present at all stages once again they illustrate the heterogeneity of osteoclasts, 40 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31–63 in this case as a function of matrix age, and the importance that osteopetrosis was due to nonfunctional osteoclasts or of understanding this phenomenon. the absence of osteoclasts, respectively (162, 163). Inter- estingly, these two groups of mice have opposing pheno- C. Osteoclast-rich osteopetrosis types with respect to bone formation. The osteoclast-rich The most frequently occurring forms of osteopetrosis c-src knockouts have increased bone formation (164), and are those caused by mutations in either the a3 subunit of the osteoclast-poor c-fos knockouts have decreased bone the V-ATPase, ClC-7, or osteopetrosis-associated transformation (165). The anabolic effects of PTH are present Ϫ Ϫ Ϫ Ϫ membrane protein 1. Osteoclasts from patients with mu- in the c-src / mice but are blunted in the c-fos / mice tations in these genes or proteins and from knockout mice (166), indicating that osteoclasts are central for bone for- have been studied quite extensively (8, 39, 56, 58, 95, mation (Table 1). 153–156). Two recent studies identified mutations in the genes for Microscopic analyses of cells from patients with defec- RANK and RANKL as the causes of osteopetrosis in a tive acid secretion by osteoclasts indicated defective ruf- novel group of patients (28, 29). No indications of osteo- fled border formation, but also accumulation of material clasts were found in these patients (28, 29), which is con- in vesicles, indicating hampered transcytosis (157). Apart sistent with previous observations in mice deficient in both from confirming the defective acid secretion and thereby RANKL and RANK (87, 90). Patients with mutations in bone resorption, when either ClC-7 or the a3 subunit is either RANKL or RANK have a pronounced osteopetrotic mutated (8, 39, 56, 58, 95, 153, 154), these studies also phenotype and classical histological hallmarks of osteo- shed light on important aspects of bone remodeling. petrosis including unresorbed primary spongiosa. How- In vitro studies indicate that osteoclasts with impaired ever, although limited data have been published, the os- acid secretion have higher survival rates than cells with teopetrotic phenotype appears to be less severe than the normal secretion, due to the reduced release of proapop- one observed in the osteoclast-rich forms (1). Thus, muta- totic signals during resorption (159). This observation tions within the RANK/RANKL/osteoprotegerin (OPG) sys- correlates well with the high numbers of osteoclasts ob- tem can lead to osteoclast-poor osteopetrosis with low bone served in vivo in this group of patients, as well as with formation in mice and men. findings in mice with attenuated acidification of the re- Interestingly, alterations in osteoblast function, such as sorption lacunae (95, 97) (Table 1). Furthermore, signif- changes in the production of RANKL and OPG, may have icantly increased resorbed areas are seen during impaired the same effect. Stabilizing osteoblastic ␤-catenin in trans- acid secretion, but the resorption pits are shallow, indi- genic mice, thus mimicking constitutive activation of the cating a disturbed activity of the osteoclasts (97). More importantly, these studies highlighted that bone forma- canonical Wnt signaling pathway, was followed by an tion in these patients is ongoing—a process that appears to up-regulation of OPG in relation to RANKL (31, 167). As be correlated to the increased number of osteoclasts rather expected, the mice developed osteoclast-poor osteopetro- than bone resorption (96, 154, 160, 161). These findings sis with failure of tooth eruption, a classical phenomenon contrast with the classical perception that bone formation in murine osteopetrosis. Mutations within LRP5 related always follows bone resorption in a tightly coordinated to the Wnt signaling pathway have underscored the fun- manner and illustrate the importance of the actual pres- damental importance of this pathway for regulation of ence of osteoclasts to maintain bone formation. bone mass. The osteoporosis pseudoglioma syndrome was found to be caused by loss of function mutations in the gene for LRP5 (168). In contrast, mutations affecting the D. Osteoclast-poor osteopetrosis Several murine forms of osteoclast-poor osteopetrosis first propeller of the coreceptor, presumed to be followed have been described in the literature (1, 42). In general, by chronic activation of the Wnt pathway, were found in whereas the mutations express a pronounced osteope- various forms of monogenic human osteosclerotic pheno- trotic phenotype and few or no osteoclasts are present, the types (169). Among these, autosomal dominant osteope- phenotypes are less severe than the phenotypes of the dif- trosis type 1 has been well characterized clinically, bio- ferent osteoclast-rich osteopetrotic mutations (1). These chemically, histomorphometrically, and biomechanically data strongly suggest that the osteoclasts are indeed in- (75). Autosomal dominant osteopetrosis type 1 is an os- volved in the production of anabolic signals for bone for- teoclast-poor osteopetrotic phenotype with increased bio- mation (3, 15). mechanical competence and no low-energy fractures. Studies of mice deficient in c-src and c-fos, a key mol- Osteoclast profiles are markedly decreased (97), bone ecule involved in ruffled border formation and a key signal formation seems to be normal, and OPG levels in the cir- transducer for osteoclastogenesis, clearly demonstrated culation increased (170). However, when investigating os- Endocrine Reviews, February 2011, 32(1):31–63 edrv.endojournals.org 41 teoclasts ex vivo from these patients, they express normal F. Other diseases characterized by increased bone resorptive capacity (30). osteoclast activity In summary, osteoclast-poor osteopetrosis can arise in Apart from hypogonadal osteoporosis, several diseases murine mutations/transgenics or humans when the OPG/ are characterized by accelerated osteoclastogenesis and RANKL/RANK system is affected directly or indirectly. function. Although the etiology of these diseases is differ- These findings underscore this cytokine system as a key ent, there are interesting overlaps and discrepancies that regulator of osteoclastogenesis. Moreover, the pheno- provide highly useful information about osteoclastic func- types seem to be less affected than the osteoclast-rich tion and secondary effects on bone formation under dif- forms, the reason for which is so far unresolved, although ferent circumstances (182). there are indications that reductions in bone formation are involved (1, 28, 29). 1. Pagetic osteoclasts Paget’s disease is a late-onset disease that is quite com- E. Pycnodysostotic osteoclasts mon in the elderly Caucasian population, where it affects An interesting subtype of osteoclasts with defective approximately 3% of individuals (182). The disease is bone resorption is observed in patients with pycnodysos- characterized by focal increases in osteoclast numbers, tosis. Pycnodysostosis is caused by loss of function or loss nuclearity, and size, which leads to localized bone destruc- of expression mutations in the cysteine proteinase cathep- tion, although surrounding osteoblasts also are activated sin K, which in humans causes dwarfism and poor bone (183) (Table 1). The identified causes of the disease in- quality due to defective remodeling of the bones (36, 171– clude mutations in four different genes, TNFRSF11A, 173). Few studies examining the phenotype of pycnodys- TNFRSF11B, VCP, and SQSTM1 (182, 184–186). These ostotic osteoclasts have been published. Microscopic anal- genes encode RANK, OPG, p97, and p62, all of which are yses of the osteoclasts have shown significantly increased involved in the regulation of osteoclastogenesis. The mu- amounts of demineralized collagen matrix in the resorp- tations all result in different subtypes of Paget’s (182, tion pit, but also inside the osteoclasts, indicating dis- 184–186). These mutations render the osteoclast precur- turbed resorption and trafficking of resorbed components sors more sensitive to RANKL stimulation, resulting in a (174, 175). A study of biochemical markers of bone turn- higher number of osteoclasts, and potentially also explain- over showed that C-terminal crosslinked telopeptide of ing the presence of giant osteoclasts (185, 187, 188). In- type I collagen (CTX-I) release was reduced, whereas pro- terestingly, a recent study in mice indicated that the most duction of the MMP-generated type I collagen fragment common mutation in p62 does not make the osteoclasts carboxyterminal telopeptide of type I collagen (ICTP) was Pagetic alone, although it sensitizes them to other yet-to- increased (67) (Table 1). Several studies of cathepsin K- be-described causes of Paget’s (189, 190). deficient mice have been published, and whereas they con- In Paget’s patients, biochemical markers of both bone firm that cathepsin K is essential for degradation of the resorption and bone formation are increased, showing an organic matrix in bone (65, 66, 176), there are also several overall increase in bone turnover at the affected sites. differences between the human and mouse phenotypes However, bone resorption clearly exceeds bone formation (103). Furthermore, in cathepsin K-deficient mice, bone (182). Whether the osteoclasts in Paget’s behave differ- formation parameters are highly increased (100). These ently from those in healthy individuals during bone re- findings have not been replicated in pycnodysostosis pa- sorption is presently unknown. In particular, it is not tients in whom the bone matrix is disordered (177), and a known whether osteoclasts in Paget’s require acidification clinical case study indicated that anabolic response to PTH to resorb bone, or whether cathepsin K is the main pro- was absent (174). Two recently published clinical studies tease, although answers to these questions might be of have shown that whereas bone resorption markers are value in the development of new therapies for Paget’s. strongly reduced, bone formation is also suppressed in Furthermore, an explanation for the localized nature of women treated with the cathepsin K inhibitor odanacatib Pagetic lesions has still not been found. Even under the (178). In a monkey study monitoring bone formation by extreme circumstances seen in Pagetic lesions, bone for- histomorphometry reductions in bone formation, rates mation is coupled to osteoclastic parameters, although were shown in the trabecular bone compartment, whereas whether this is due to increased osteoclast numbers or bone formation was increased in the cortical compartment activities is not known. Moreover, in this case a treatment (179–181). type eliminating the activity of both types of cells is most In conclusion, cathepsin K mediates cleavage of type I likely to be preferred because the increase in bone forma- collagen in the resorption lacunae, but its secondary ef- tion occurring is part of the pathology, and likely will fects on bone formation are bone type-dependent and still provide no benefit for the bones if maintained. Thus, need to be investigated further. bisphosphonate, which strongly attenuated overall bone 42 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31–63 turnover, appears to be highly relevant in the context of 3. Arthritic osteoclasts Paget’s disease (191). Later stages of RA are characterized by massive bone destruction caused by osteoclasts (212, 213). However, 2. Osteolytic osteoclasts there are several indications that these osteoclasts are not Several forms of cancer can metastasize to bone and classical bone-resorbing osteoclasts but include cells that form osteolytic metastases (192–196). Once the cancer degrade calcified cartilage (83, 214) (Table 1). Several has reached the bone, tumor and bone interact in a vicious studies have indicated that TNF-␣ at least partially drives cycle in which tumor-secreted factors, such as PTHrP, osteoclastogenesis in RA (215), as exemplified by mice stimulate bone cells, which in turn release growth factors overexpressing human TNF-␣ with massive joint destruc- and cytokines that promote further tumor cell growth tion including bone erosion (216). Furthermore, TNF-␣- (192, 197). The activation of osteoclastogenesis induced neutralizing antibodies, such as infliximab, or soluble by tumor cells has been shown to involve a switch in the TNF-␣ receptor antagonists, such as etanercept, provide RANKL/OPG ratio favoring osteoclastogenesis and acti- amelioration of RA in humans (217, 218). Apart from vation, leading to release of the tumorigenic factor TGF-␤, TNF-␣, RANKL is, not surprisingly, a crucial factor in and thereby inducing the vicious cycle (198). As a function osteoclastogenesis during RA (87), and mice deficient in of the increased numbers of osteoclasts and accelerated RANKL are protected against bone, but not cartilage, ero- bone resorption, a marked up-regulation of osteoblast ac- sion (219). Treatment with OPG of mice with collagen- tivities is also observed (199, 200). induced arthritis also leads to amelioration of bone de- The activity of osteoclasts in metastases has been mon- struction, while having a markedly lower effect on itored closely using biochemical markers of bone turnover cartilage degradation (220). In addition, a study in which (199, 200), and these studies have indicated that bone TNF-␣ overexpressing mice were crossed with mice defi- resorption by tumor-induced osteoclasts to some extent cient in c-fos (i.e., deficient in osteoclasts), showed no bone depends on MMP activity, rather than cathepsin K, because the type I collagen fragment ICTP is released in high destruction but clear evidence of cartilage destruction amounts (199, 201) (Table 1). Animal models of breast (221). Furthermore, human clinical trials using deno- cancer bone metastases are to some extent sensitive to sumab have demonstrated that the RANKL/RANK axis is inhibitors of both cathepsin K and MMPs (101, 202–204); a key player in RA and that inhibition of RANKL signaling however, clinical data for MMP inhibitors have been dis- may provide a useful treatment option (222). Finally, ␣ ␤ appointing (205, 206). An interesting question is whether more recent evidence has indicated that IL-1 and IL-1 these agents, to be effective, have to inhibit MMPs before the both play a partial role in bone resorption and cartilage tumors actually metastasize. For cathepsin K inhibitors, the degradation (223). Anakinra, which is a soluble IL-1 data indicate a beneficial effect on the release of the bone receptor antagonist, is also used for treatment of RA, ␣ resorption marker N-terminal crosslinked peptide of type although it appears to be less effective than the TNF- I collagen, and an increase in ICTP levels (204). However, inhibitors (217). In addition, tocilizumab (anti-inter- further information is needed to draw reliable conclusions leukin-6 receptor inhibitor) has shown promise in pre- on the usefulness of cathepsin K inhibitors for metastatic venting RA progression through an effect including a bone disease. reduction in osteoclast numbers (158, 324). In contrast, treatments ablating both osteoclasts and Because osteoclasts play a significant role in RA, the increased osteoblast activity, such as denosumab and bisphosphonates appear to be an attractive treatment op- bisphosphonates, reduce the destructive capacity of the tion. Early evidence has indicated that zolendronate may metastasis and, importantly, the afflicted pain. How- be useful (224), although this has not been fully estab- ever, they do not appear to affect the cancer cells (192, lished yet (225). Furthermore, interpretation of the effects 207–210), although there are some indications that the of bisphosphonates in RA is often clouded by glucocorti- bisphosphonates affect the life span of the cancer cells coid treatment of the same patients because glucocorti- as well as reducing osteolysis (211). coids are associated with rapid systemic bone loss, In summary, from a treatment point of view, there are independent of RA (226, 227). However, in both collagen- several similarities between Paget’s and osteolytic metas- induced arthritis in rats and in the TNF-␣ transgenic tases. The optimal approach appears to involve a strategy mouse model, zolendronate was effective in reducing both of reducing overall bone turnover toward the normal bone and cartilage destruction (228, 229). range, such as with the use of denosumab or bisphospho- The bone resorption process in RA is still not com- nates An intriguing possibility would be to target only the pletely understood despite several studies into the molec- areas undergoing destruction, but whether this is feasible ular mechanisms. The role of cathepsin K has been exten- is presently not known. sively studied, and the data are somewhat conflicting Endocrine Reviews, February 2011, 32(1):31–63 edrv.endojournals.org 43

(230–232). Overexpression of cathepsin K has been important secondary role in bone remodeling, which is of shown to accelerate joint destruction in mice (231), and importance when developing novel treatments for osteo- overexpression of cathepsin K has been observed in hu- porosis (15). Second, excessive and local activation of mans with RA (233, 234). However, studies in the TNF-␣ osteoclasts occurs in several diseases, and interestingly overexpression model crossed with cathepsin K-deficient the osteoclasts appear to switch subtype with respect to mice showed that cathepsin K plays only a marginal role their resorption machinery. These findings highlight the in bone resorption in RA (232), a finding supported by a importance of characterizing the function of osteoclasts case study showing severe arthritis in a pycnodysostotic under pathological circumstances to optimize treatment patient (235), although there are still controversies with strategies. respect to the role of cathepsin K in RA (236). Other cathepsins have not been explored in detail, and their expression patterns do not indicate a particular effect VI. Drug-Induced Osteoclast Subtypes on osteoclast function in RA (237). Under some circumstances, MMPs also play a role in A. Existing drugs bone resorption (2, 81). Studies showing that the MMP- Several antiresorptive drugs for the treatment of osteo- derived collagen type I fragment, ICTP, is increased in RA porosis, as well as glucocorticoids and PTH treatment, are could indicate that osteoclasts used MMPs to digest ma- known to alter osteoclasts in various ways. These drugs all trix under these circumstances (238, 239). Infliximab provide critical information on osteoclast function, and treatment has been shown to reduce ICTP levels, as well as furthermore, they have also played a great role in illus- osteoclast numbers (240), further indicating that oste- trating the interplay between osteoclasts and osteoblasts, oclasts utilize MMP-mediated bone degradation in RA. as will be described in the following section. However, a direct link between the production of ICTP and osteoclasts has not been demonstrated yet. 1. Bisphosphonates Whether acid secretion by osteoclasts is needed for Bisphosphonates have long been associated with induc- bone destruction in RA is also not clear. Because bone tion of apoptosis in osteoclasts, and the mechanism of destruction is likely to occur as a result of MMP activity, action underlying the apoptotic effect depends on whether the need for acidification may be reduced when compared or not the bisphosphonates contain nitrogen (242). Both with “classical” bone resorption (69), although this is still classes of bisphosphonates bind to the bone matrix and are not fully understood. Another possibility is that MMP- taken up by the osteoclast during bone resorption. The mediated collagen type I degradation is mediated by an- simple bisphosphonates are metabolized into toxic ATP other cell type, although this still remains to be clarified. analogs, thereby inducing osteoclast apoptosis in vitro A case study of arthritis in a case of autosomal dominant (242). The nitrogen-containing bisphosphonates exert osteopetrosis type II (241) showed a lack of bone degra- their function by inhibiting the mevalonate pathway, dation, whereas cartilage degradation was abundant, which leads to the generation of an ATP analog known to thereby mimicking the situation seen in osteoclast-defi- induce apoptosis in osteoclasts in vitro (242). The antire- cient systems (221) and indicating that bone resorption in sorptive potency of the nitrogen-containing bisphospho- RA depends fully on acid secretion. nates in vivo is controlled by mineral binding affinity and In summary, development of severe RA involves oste- by their ability to inhibit the mevalonate pathway (242). oclasts, and a reduction of bone resorption by these cells Although in vitro data clearly show that bisphospho- is desired. This may be obtained through inhibition of nates induce apoptosis, analyses of osteoclast numbers in inflammation and thereby bone and cartilage destruction, iliac crest biopsies failed to show a reduction in the number as seen with anti-TNF-␣ therapy. Alternatively, therapies of osteoclasts when patients were treated with bisphos- such as denosumab that target the osteoclasts directly may phonates (243–245). On the other hand, bisphosphonates also be useful, although these fail to eliminate inflamma- reduce systemic levels of TRACP 5b and cathepsin K, both tion and only partially prevent cartilage degradation markers of osteoclast number (32, 246–248), potentially (220). The optimal therapy could be a combination of indicating that osteoclasts undergo systemic apoptosis, antiinflammatory and antiosteoclastic measures, al- which correlates well with the expected effects of bisphos- though this is presently not known. phonates (242) (Table 1). Other studies have shown that In summary, studies of osteoclasts under pathological when bisphosphonate therapy continues for more than 1 circumstances have highlighted some important phenom- yr, the number of circulating osteoclast precursors is re- ena. First, osteoclasts themselves, not just their resorptive duced, and these reductions are speculated to be related to activity, mediate bone formation and therefore perform an reduced serum RANKL levels (249, 250). 44 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31–63

A recent study of biopsies from alendronate-treated pa- a. Osteonecrosis of the jaw (ONJ) and bisphosphonates. tients showed the presence of giant hypernucleated, de- Bisphosphonate therapy, especially in the case of malig- tached, and frequently apoptotic osteoclasts, and the num- nancy-induced bone loss, has been connected to the oc- ber of these abnormal osteoclasts correlated with the currence of ONJ, mainly due to the ability of bisphospho- cumulative dose of bisphosphonate (251). Although in- nates to strongly suppress bone turnover (260–262). teresting, the biological implications of this finding are not Although the probability of ONJ is very low for the dosing clear yet. regimens used for treatment of osteoporosis, there has still One potential explanation for the discrepancies in scor- been a lot of debate about whether ONJ is the result of the ing osteoclasts in the iliac crest biopsies is the very low massive suppression of bone turnover in the jaw (262). number of osteoclasts observed in general. Recent reports Interestingly, alveolar bone of the jaw is very similar to have also debated the clinical relevance of studying iliac bone matrix in the long bone, i.e., it contains the classical crest biopsies because they are from non-weight-bearing cell types as well as the lamellar structure (263). Further- bones and these are different from weight-bearing bones more, bone remodeling occurs normally in alveolar bone, (252–254), and in general more data are needed to draw although the rate of remodeling has been estimated to be a final conclusion on the osteoclastic response to up to 10-fold higher than the corresponding rate in long bisphosphonates. bones (263–266). In ONJ, the number of osteoclasts has On the other hand, the effect on reduction of bone re- been investigated, and it appears that the osteoclasts are sorption measured both by biochemical markers and by absent from the lesions (267, 268), although opposing evi- bone histomorphometry (activation frequency) confirms a dence also exists (269) and thus more studies are needed. potent reduction in bone resorption, and the level of It has been speculated that massive suppression of os- reduction is often down to the lower range of premeno- teoclast function, and thus bone turnover, in this high- pausal levels, although this depends heavily on the ef- turnover compartment is what causes ONJ to occur; how- ficacy of the individual bisphosphonate (242, 243, 245, ever, there are several other factors involved, such as tooth 255–258). extraction or infections, and the overall causality is still With respect to secondary effects on bone formation, not clear (262). One point of particular interest is measurement of biochemical markers of bone turnover whether this phenomenon is specific for bisphosphonates or whether it will happen with other very potent shows a marked reduction in bone formation markers, and long-lived antiresorptives; however, this is pres- and the effects are maintained throughout the treatment ently not known. period, although this again is dependent on the individual bisphosphonate (242, 243, 245, 255–258). Biopsy studies 2. Selective estrogen receptor modulators/hormone have confirmed that bone formation is reduced when com- replacement therapy pared with placebo, and although the reduction in bone Because cessation of estrogen production is a major formation rates is dependent on the individual bisphos- cause of osteoporosis (3, 14) and both estrogen and phonate, the data indicate that bone formation is not com- SERMs are used for treatment of osteoporosis, several pletely suppressed but is reduced to the lower postmeno- studies have been conducted to clarify their effect on pausal levels (243, 245, 258). The FLEX study (Fracture osteoclasts. Intervention Trial Long-term Extension), although show- Estrogen has been shown to exert direct antiosteoclas- ing continued reductions in vertebral fractures, increase in tic effects at several stages of osteoclastic differentiation bone mineral density (BMD), and reduction of bone turn- and function, namely osteoclastogenesis, resorption, and over markers with alendronate, did not show a significant apoptosis. Direct inhibition of the formation of multinu- reduction in bone formation rates when comparing pa- cleated osteoclasts is thought to be caused by suppression tients stopping alendronate to patients continuing treat- of RANKL-induced c-Jun and basal c-Jun N-terminal ki- ment; however, the numbers of biopsies were low (259). nase activity in osteoclast precursor cells (135, 136). In All in all, there is no doubt about the fracture-prevent- nonpurified osteoclast-precursor systems, estrogen was ing effects of bisphosphonates; however, knowledge of the found to inhibit osteoclastic differentiation in a human ␣ ␤ effect of bisphosphonates on osteoclasts in vivo is quite system (270), possibly via down-regulation of the v 3 limited. Apoptosis of the osteoclasts most likely explains integrin (271). Two studies of estrogen have been con- the reduction in bone resorption. Furthermore, although ducted using CD14ϩ osteoclast precursors. As mentioned the extent of the secondary reduction in bone formation is earlier, one study showed significant inhibition of oste- still discussed, it appears to be clinically relevant, and it oclastogenesis (137), whereas the other showed no direct most likely is the explanation for the attenuation of the effect on osteoclast precursors (272). To date, there is no BMD increase seen after the first year of treatment. explanation for this discrepancy. Endocrine Reviews, February 2011, 32(1):31–63 edrv.endojournals.org 45

Studies of the effects of SERMs on osteoclasts have vertebral fractures, most likely due to low exposure, the shown that tamoxifen inhibits osteoclastogenesis directly, clinical usefulness is limited (122). Recent studies have whereas raloxifene and ospemifene only inhibited osteo- indicated that a recently developed oral formulation of clasts through up-regulation of the expression of OPG by salmon calcitonin will lead to improved efficacy because it osteoblasts (273). Although early studies showed an effect has been optimized with respect to pharmacokinetic and of raloxifene on osteoclastogenesis, these were conducted pharmacodynamic properties. This has led to a 10-fold using mixed cell populations and therefore most likely higher exposure and thereby a greater reduction in bone reflect the increase in OPG (274). resorption parameters. Thus, this agent will most likely Mature osteoclasts have also been shown to respond provide improved efficacy in preventing fractures (291), directly to estrogen (275, 276). These studies showed that and although it remains to be proven in long-term clinical both the activity and the production of the lysosomal en- trials, the phase II data are promising (127). zymes are down-regulated by estrogen (277, 278), possi- The mode of action of oral calcitonin is a transient bly explaining the reduction in resorption by the down- suppression of the nocturnal rise in bone resorption ob- regulation of cathepsin K and TRACP (36, 66, 77, 279) tained by giving the treatment at the right time of day—in (Table 1). the evening (292), which results in a reduction in bone In summary, in vitro data clearly demonstrate that es- resorption, but no effect or very modest secondary effects trogen and SERMs reduce osteoclast numbers via inhibi- on bone formation (127) (Table 1). These findings are tion of osteoclastogenesis, and potential effects on bone further supported by other clinical studies showing that resorption and apoptosis might add to the in vivo effect. calcitonin may inhibit bone resorption without affecting Although some studies of osteoclasts in patients treated bone formation, a finding observed independent of ad- with either HRT or SERMs have been conducted, the ef- ministration route (293–296). fects of both estrogen and SERMs on bone remodeling There are histological indications that calcitonin atten- indices based on histomorphometry are quite modest uates ruffled border formation by osteoclasts (296–298), (280–284). Overall, these studies show a reduction in ac- and this appears to be the mode of action underlying the tivation, frequency, and depth of resorption, as well as— antiresorptive effects of calcitonin in vivo, thereby elabo- where detectable—a small decrease in osteoclast numbers. rating on the previously described transient reduction in Reduced bone formation rates were also observed, con- bone resorption (292). firming the coupled nature of inhibition mediated by es- Studies of mice lacking the calcitonin receptor indicated trogen and SERMs (280–284). These data are corrobo- that bone formation was increased, and thus that calcito- rated by biochemical markers of bone turnover, which nin is a suppressor of bone formation (299, 300). These clearly demonstrated a coupled reduction in bone resorp- studies were conducted mainly in young mice. A recent tion and bone formation (32, 285–287), and furthermore study in mice deficient in the calcitonin receptor specifi- explain the plateau effect observed in BMD measurements cally in osteoclasts failed to reproduce this finding (301, after 1 yr (285). 302). However, considering the very modest, or nonex- In summary, many of the numerous studies of the in istent, suppression of bone formation in patients treated vitro mode of action of HRT and SERMs show a reduction with calcitonin, the mice data appear of low relevance in in osteoclastogenesis. In alignment, in vivo studies of these the clinical setting (127, 293–296). therapies on osteoclasts confirm that osteoclastogenesis is Further studies are needed to understand this potential lower than in the untreated population, and importantly, dissociation of bone resorption and bone formation. It these also confirm the secondary decrease in bone may be that this dissociation occurs because calcitonin formation. disappears quickly from the circulation and thus is a completely reversible treatment (122). An interesting question 3. Calcitonin is whether calcitonin treatment may result in better bone Calcitonin is a natural peptide hormone produced by quality than potent antiresorptives due to the lack of effect parafollicular cells (C cells) of the thyroid gland. Calcito- on bone formation and the lower suppression of bone nin possesses potent antiresorptive effects (288), and bind- resorption, which is expected to lead to a slow, yet pro- ing of calcitonin to the calcitonin receptor on osteoclasts longed increase in BMD (6, 303). induces a rapid change in the cytoskeletal structure of the osteoclasts in vitro, which in turn leads to a reduction in 4. Parathyroid hormone bone resorption without inducing apoptosis of the cells Although PTH does not appear to affect osteoclasts (102, 289, 290). Calcitonin in either an injectable or a directly because these cells do not appear to express the nasal form has been approved for treatment of osteopo- PTH receptor, PTH nonetheless affects osteoclast func- rosis; however, because it only prevents about 35% of tion on many different levels (10). In vitro studies of the 46 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31–63 effects of PTH on osteoclasts all show that PTH induces Furthermore, strontium has also been shown to increase osteoclastogenesis and that induction of a transient OPG expression by osteoblasts (330). RANKL expression is essential for this effect (10). How- In summary, strontium ranelate is a very interesting ever, PTH has mainly been studied in relation to its pow- molecule with respect to effects on osteoclasts, and several erful anabolic effects on osteoblasts (10). Intermittent dos- lines of in vitro evidence indicate that it reduces osteoclast ing of PTH in human subjects results in a marked increase function (326). However, the relevance of the effect on in bone formation markers, and secondarily in activation osteoclasts is still debated, and thus the overall effects of of bone resorption through increased RANKL expression this “uncoupling” molecule are still not fully understood. (9, 304, 305). Bone histomorphometric and biochemical 6. Glucocorticoids marker studies confirm the increase in bone turnover Glucocorticoids are used to overcome inflammatory (306–308) (Table 1). conditions, such as inflammatory bowel diseases and RA The anabolic mode of action of PTH has been debated (331). Glucocorticoid use is associated with severe bone extensively. Studies show that PTH directly activates bone loss due to strongly attenuated bone formation (332). This formation by osteoblasts when given intermittently (309, attenuation of bone formation leads to a rapid accelera- 310). In mouse models that are either deficient in osteo- tion in the number of fractures in glucocorticoid-treated clasts or deficient in bone resorption, data suggest that the patients (332), especially in trabecular bone compart- anabolic effect of PTH is dependent on the presence of ments such as vertebrae (333). Glucocorticoid treatment is mature osteoclasts, but not on their activity (165, 166, the most common cause of secondary osteoporosis (333), 311). Furthermore, initial clinical trials combining alen- and thus patients on glucocorticoids are often treated with dronate and PTH showed that alendronate blunted the antiresorptives (334). anabolic effect of PTH (312, 313), and there were indi- In vivo, glucocorticoids inhibit osteoblastogenesis, the cations that even pretreatment with alendronate led to a generation of bone-forming osteoblasts, and promote ap- blunting of the PTH response (314). On the other hand, optosis of osteoblasts and osteocytes, which is consistent animal studies indicate that PTH can be combined with a with the well-known inhibition of bone formation (335). bisphosphonate (315, 316), but, as noted by Johnston et In contrast, the cellular effects of glucocorticoids on al. (315), there are marked differences in the doses of PTH osteoclasts are a subject of controversy. In vivo, the effects used in rodents and in humans. appear to fall into two categories, one being a short-lived Collectively, PTH exerts marked regulation of bone acceleration of osteoclastogenesis and bone resorption, turnover (15), including the activation of osteoclasts. The whereas the other is a reduction in osteoclast numbers, potential anabolic role of osteoclasts and, especially, how which is not well-characterized with respect to exposure to achieve the right osteoclast subtype are debated in- time to glucocorticoids (335–340) (Table 1). Interestingly, tensely. Future studies will most likely explain this com- a study by Kim et al. (340) showed that the detrimental plex interplay between bone cells and thus guide the right effect of glucocorticoids on bone formation was absent combination of PTH and antiresorptive. when the glucocorticoid receptor was ablated specifically in osteoclasts in mice. 5. Strontium ranelate In vitro studies of glucocorticoids are often conducted Strontium ranelate is approved for treatment of osteo- in the presence of contaminating cells, and because glu- porosis, albeit only in Europe, through its ability to reduce cocorticoid treatment also promotes RANKL and reduces fracture risk in patients (317–321). The mode of action has OPG expression in osteoblasts, it is unclear exactly to been studied extensively, and yet it is not fully clear exactly what extent they influence the osteoclasts (341, 342). Two how it works in vivo. Bone biopsies have been investi- recent studies showed that glucocorticoid treatment hy- gated, and these indicated small increases in bone forma- peractivated osteoclasts and thus suggests that glucocor- tion and mineralization rates but no changes in bone re- ticoids indeed have a direct effect on bone resorption (343, sorption or osteoclast parameters, thus indicating that 344). Yet some studies show the opposite (340). Overall, strontium ranelate stimulates novel bone formation (322). the results appear to be very context-dependent, illus- These data were supported by analysis of biochemical trating the complex nature of the biological effects of markers of bone turnover demonstrating increased bone glucocorticoids. formation (308, 323), while also showing a modest de- Measurements of biochemical markers of bone turn- crease in bone resorption markers (323, 325). over in human subjects on glucocorticoid therapy pro- In vitro studies support the hypothesis that strontium vided diverse results, which appeared to be dependent on ranelate has a dual effect, namely inhibition of bone re- the dose of glucocorticoid used (331, 345, 346). However, sorption while stimulating bone formation (326–329). biochemical marker data indicate that bone resorption Endocrine Reviews, February 2011, 32(1):31–63 edrv.endojournals.org 47 increases short term, whereas bone formation is attenu- resorption by osteoclasts (36, 65, 66). Studies conducted ated long term (345) in response to glucocorticoid ther- in pycnodysostosis patients before the final identification apy, which corresponds well to mouse studies (335–339). of cathepsin K showed massive accumulation of nondi- The short-term increase in bone resorption and long-term gested bone collagen fibers in the resorption pit below the suppression of bone formation are also observed with his- osteoclasts (175). These findings were matched by those tomorphometry in mice (347, 348). from investigations in cathepsin K-deficient mice (66), In summary, glucocorticoids exert detrimental effects demonstrating a critical role for cathepsin K in degrada- on bone, and whereas the effects on osteoclasts are not tion of the organic matrix. Further studies in cathepsin completely clear yet, further investigation of the effect on K-deficient systems have indicated that cells of the osteo- the coupling between osteoclasts and osteoblasts could blast lineage, namely bone-lining cells (68); cells of hema- explain the overall beneficial effect of antiresorptives on a topoietic origin (69); and a general up-regulation of the syndrome mediated primarily by suppressed bone forma- osteoclastic stimuli, osteoclast numbers, and proteases, tion (227, 349). These findings further highlight the im- especially RANKL and MMPs (103), are involved in com- portance of understanding the interplay between bone pensating for the lack of cathepsin K. Interestingly, a hall- cells to provide the optimal treatment. mark of the absence of cathepsin K function is the presence of the MMP-derived collagen fragment ICTP, which is B. Future treatments seen in pycnodysostosis patients, cathepsin K-deficient A series of interesting targets for osteoporosis treat- mice, and cell cultures (67, 69, 103, 356), strongly indi- ment are currently under investigation. The targets of cating a compensation by MMPs in the absence of cathep- these treatments to some extent employ novel modes sin K (Table 1). of action on osteoclasts. These novel modes of action are An interesting study by Fuller et al. (357) showed that of importance when investigating whether they may have inhibition of cathepsin K in cultured osteoclasts led to secondary effects on bone formation, and subsequently on augmented secretion of IGF-I. Furthermore, increased bone quality. numbers of osteoclasts, containing granules of matrix proteins, have been observed in monkey studies of cathepsin 1. Denosumab K inhibitors (175, 181), thus indicating the potential of Denosumab is a fully humanized monoclonal antibody this protease for anabolic stimulation of the osteoblasts. to RANKL; it has gone through a phase III fracture effi- Cathepsin K-deficient mice have been studied extensively, cacy trial in which it was shown to reduce fracture rates by and recent experiments indicate that bone formation in 68% in vertebrae and 40% in hip (351); and it was re- trabecular bone is increased after cathepsin K administra- cently accepted for treatment of severe osteoporosis in both the United States and Europe. tion and thus that bone resorption and bone formation are In line with in vitro studies of inhibition of RANKL not coupled (100, 176). However, clinical studies of ca- (210, 352), denosumab prevents osteoclastogenesis, thepsin K inhibitors, such as odanacatib, have shown that blocks bone resorption, and increases osteoclast apopto- whereas a robust reduction in CTX and N-terminal sis. It induces a massive reduction of osteoclasts in vivo crosslinked peptide of type I collagen occurred and no and, thereby, almost complete suppression of bone resorp- changes in TRACP 5b were observed, a significant de- tion in both humans and mice (352–354) (Table 1). De- crease in the bone formation marker pro-peptide of col- nosumab treatment also leads to a marked suppression of lagen type I and nonsignificant reductions in bone forma- bone formation markers in humans (353, 354), as well as tion rates by histomorphometry were seen (178). a marked suppression in bone formation rates measured Furthermore, a study of osteoclast morphology as a func- by histomorphometry in animal models (352, 355). Thus, tion of cathepsin K inhibition in humans indicated in- denosumab treatment is consistent with the classical per- creased size of the osteoclasts and the presence of large ception of coupling. TRACP-positive vacuoles, yet no increase in osteoclast A key point with respect to denosumab is whether the numbers (358). Studies in monkeys clearly demonstrated suppression is too severe and could lead to detrimental that bone formation in the trabecular compartments was effects on bone quality long term (6). However, as is the dose-dependent and significantly reduced by cathepsin K case with bisphosphonate treatment, this is not clear at inhibitors, whereas an induction of bone formation was present. observed at cortical sites (179, 180). Further studies are needed to clarify whether the osteoclasts in cathepsin K- 2. Cathepsin K inhibitors deficient situations indeed signal to the osteoblasts. An Cathepsin K is a critical protease for degradation of the indication came from a pycnodysostosis case study that type I collagen matrix in the resorption pits during bone showed no bone formation response to PTH (174), and 48 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31–63 thus indicated that secretion of the coupling signals may be and showing no inhibition of bone formation markers (8, attenuated at least in human systems. A possible expla- 364). Similar findings were published for an inhibitor of nation for the lack of secondary anabolic effects induced the V-ATPase (365). In a study of prosthetic implants by inhibition of cathepsin K is the presence of demineral- coated with bafilomycin, osteoclast numbers were ele- ized collagen fibers in the resorption pit, which are re- vated, and indications of increased bone formation were moved by bone-lining cells (68). Although it is not well observed (366). These studies were the first to provide understood how the presence of fibers and their subse- proof of concept that inhibition of acidification is a really quent removal affect osteoblasts, a study indicated that promising target for osteoporosis treatment. Most inter- RGD sequences, which are numerous in collagen, antag- estingly, bone formation levels, as measured by osteocal- onize osteoblast function (359). cin and by evaluation of the dynamic histomorphometry These findings again illustrate the importance of care- parameters mineral apposition rate and the mineralizing fully investigating the osteoclast subtype as a function of surface vs. bone surface, were not affected. These data cathepsin K inhibition to more accurately predict potential therefore suggest that inhibition of acidification of the secondary effects on bone formation. osteoclastic resorption lacunae results in an uncoupling of bone formation and bone resorption, thereby possibly im- 3. Glucagon-like peptide-2 proving the potential efficacy of the treatment. This is in GLP-2 is a 33-amino acid peptide. GLP-2 is created by contrast to other antiresorptive treatments where a sec- specific posttranslational proteolytic cleavage of proglu- ondary decrease in bone formation is observed (3, 367). cagon in a process that also liberates the related GLP-1 These data also indicate that the subtype of osteoclasts (124). GLP-2 is produced by the intestinal endocrine L cell obtained—nonresorbing yet alive—when targeting acid and by various neurons in the central nervous system secretion is active with respect to bone formation, and thus (124). Intestinal GLP-2 is cosecreted along with GLP-1 might possibly be combined with PTH treatment in the upon nutrient ingestion. future. GLP-2 has in clinical settings been demonstrated to in- Finally, other compounds that appear to modulate the hibit bone resorption (124–126) (Table 1). Reductions in activity of osteoclasts are in development for osteoporosis. bone resorption by exogenous GLP-2 require an intact These include calcilytics, PTHrP, and sclerostin, but their gastrointestinal tract (125, 361, 362). The decreased meal- effects on osteoclasts, which most likely are indirect, are induced inhibition of bone resorption in jejunostomy pa- not clear yet (368–370), and thus these will not be de- tients, who lack a GLP-2 response, supports the view that scribed further. GLP-2 plays a role in postprandial reduction in bone resorption (361, 362). GLP-2 has in addition been suggested to inhibit bone VII. The Bone Anabolic Effects of resorption without affecting bone formation (125), high- the Osteoclasts lighting this mode of inhibition of resorption for further Since the early discovery that osteoclast activities were investigation with respect to osteoclast subtypes. involved in regulation of bone formation during targeted remodeling (17, 18, 371, 372), a series of studies have 4. Acid secretion inhibitors investigated the nature of this process. Acid secretion by osteoclasts has been an interesting The early studies focused mainly on the release of mol- therapeutic target since the discovery that this process is ecules from the bone matrix during bone resorption and controlled by the a3 subunit of the V-ATPase and ClC-7, identified molecules such as IGF-I and TGF-␤ (357, 373, both of which are quite specific to osteoclasts (37, 39, 56). 374). However, with the recent discovery that mature os- Furthermore, in vitro studies of osteoclasts treated with teoclasts, not osteoclast precursors and not necessarily inhibitors of these ion transporters have shown that the bone resorption, are needed for stimulation of bone for- osteoclasts are unable to resorb bone and that they there- mation (8, 9), a series of studies have investigated this fore survive longer (8, 159, 363), thereby mimicking the phenomenon. elevated numbers of osteoclasts observed in patients with Zhao et al. (12) demonstrated that osteoclast-mediated mutations in the genes for a3 and ClC-7 (37, 97) (Table 1). expression of EphrinB2 and osteoblast-mediated expres- In aged ovariectomized rats, early low-potency chloride sion of EphB4 were involved in a bidirectional communi- channel inhibitors were able to prevent bone resorption by cation between these cell types. EphrinB2 on osteoclasts approximately 50%, as monitored by both BMD and the stimulated bone formation by the osteoblasts via binding biochemical markers of bone resorption CTX-I or deoxy- to EphB4, while EphB4 expression on osteoblasts in turn pyridinoline, while augmenting the number of osteoclasts inhibited osteoclastogenesis via binding to EphrinB2 (12). Endocrine Reviews, February 2011, 32(1):31–63 edrv.endojournals.org 49

However, ephrin signaling requires close contact between gies; and 2) stimulation of bone formation by the osteo- the osteoclasts and their target cells. This has led to the blasts, a process that as illustrated by studies conducted in speculation that ephrin signaling could be involved in the osteopetrotic patients is, to a large extent, independent of interplay between osteoclasts and bone-lining cells, which bone resorption. It is presently not completely clear when are found in close contact and appear to regulate the ac- the osteoclasts are anabolically active, yet it appears to be tivity of each other (68, 375). related to the presence of large multinuclear osteoclasts Stimulatory signals from osteoclasts directly to mature because bone anabolic responses are seen under these cir- bone-forming osteoblasts are, on the other hand, likely to cumstances (3, 15). be paracrine because these cell types are not found in close Understanding osteoclast functioning may be useful for contact (264). Both TGF-␤ and IGF-I are produced by the developing drugs that not only inhibit bone resorption but osteoclasts and are known to stimulate bone formation also enable bone resorption levels that ensure targeted under various circumstances (376–380). In relation to remodeling and, importantly, support continued anabolic these findings, it is interesting that the anabolic effect of signaling from osteoclasts to osteoblasts in the bone re- PTH in mice was shown to be mediated through IGF-I modeling compartment. This has the triple effect of: (350), an effect that is absent in the absence of osteoclasts 1) maintaining a sufficient resorption level and thereby (166). This confirms that IGF-I is a coupling factor. avoiding excessive aging of the bones; 2) sustaining a local A recent study demonstrated the mature human osteo- stimulation of bone formation at the resorption site only; clasts, independent of their resorptive activity, secrete fac- and 3) not initiating induction of bone formation in oth- tors that activate nodule formation by the osteoblasts (11). erwise quiescent sites. Theoretically, this type of inhibition This study was followed by a study showing that oste- of bone resorption would allow a continuous, ongoing oclasts produce the anabolic factors bone morphogenetic increase in BMD, which is in contrast to the effects of the protein 6, Wnt10b, and sphingosine-1-phosphate, again presently approved antiresorptives where a plateau effect independent of bone resorption (13). Furthermore, inhi- on BMD is observed within the first 12–18 months. This bition of bone morphogenetic protein 6, Wnt10b, and means that even with less powerful suppression of bone sphingosine-1-phosphate led to inhibition of the oste- resorption, such as that seen with the oral formulation of oclast-mediated stimulation of bone formation in vitro. salmon calcitonin, the long-term effects would surpass Finally, osteoclasts have also been shown to produce car- those of the bisphosphonates. diotrophin-1 (CT-1), which activates bone formation by A deeper understanding of both the differences in the osteoblasts, although the role of CT-1 was clearly shown resorption process depending on circumstances and the to be dependent on age because loss of CT-1 in newborn knowledge relating to when the osteoclasts are anaboli- mice caused osteopenia, whereas in larger mice it caused cally active will aid in the identification of novel treatment mild osteopetrosis due to defective bone resorption (360). opportunities for bone diseases. In summary, the presence of mature osteoclasts is associated with the secretion of stimulation of bone anabolic Finally, the use of biochemical markers of bone turn- signals, and whereas several candidate factors have been over is becoming increasingly relevant for the continued identified, a clear demonstration that removal of one of the understanding of osteoclasts. Markers provide systemic molecules specifically in the osteoclasts in vivo leads to loss information on the outcome of a given treatment and can of bone formation is still missing. help answer questions such as whether glucocorticoids exert detrimental effects on bone formation, and whether antiresorptives antagonize bone formation secondary to VIII. Conclusions and Future Perspectives bone resorption because of suppression of osteoclast numbers or activity. Osteoclasts have traditionally been viewed as bone resorption “machines”; however, studies of osteoclasts have highlighted that these cells are highly context-specific, and the context of the individual osteoclasts is important for Acknowledgments the continued regulation of bone remodeling. The Danish Research Foundation “Den Danske Forskningsfond” is ac- As described in detail in this review, the osteoclasts knowledged for financial support. possess at least two highly important functions: 1) bone resorption, a process that is highly dependent on a series Address all correspondence and requests for reprints to: K. Henriksen, of external stimuli, such as matrix type, remodeling status, Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Den- mark. E-mail: [email protected]. hormones involved in calcium homeostasis, genotype, in- Disclosure Summary: M.A.K. owns stocks in Nordic Bioscience A/S. flammation, and importantly also on intervention strate- All other authors have no conflicts of interest. 50 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31–63

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Cellular mechanisms of bone remodeling

Erik Fink Eriksen

Published online: 29 December 2010 # The Author(s) 2010. This article is published with open access at Springerlink.com

Abstract Bone remodeling is a tightly regulated process (RUNX, Osterix) are involved in osteoblast differentiation. securing repair of microdamage (targeted remodeling) and Both pathways are modulated by calcitropic hormones. replacement of old bone with new bone through sequential osteoclastic resorption and osteoblastic bone formation. The Keywords Osteoblasts . Osteoclasts . Lining cells . Growth rate of remodeling is regulated by a wide variety of factors . Cytokines . Bone remodeling . Osteoporosis . Bone calcitropic hormones (PTH, thyroid hormone, sex steroids remodeling compartment etc.). In recent years we have come to appreciate that bone remodeling proceeds in a specialized vascular structure,— the Bone Remodeling Compartment (BRC). The outer 1 Introduction lining of this compartment is made up of flattened cells, displaying all the characteristics of lining cells in bone Bone histomorphometry has given us great insights into including expression of OPG and RANKL. Reduced bone bone physiology and bone remodeling in particular. turnover leads to a decrease in the number of BRCs, while Histomorphometric indices obtained using tetracycline increased turnover causes an increase in the number of double labeling techniques are unique, because they BRCs. The secretion of regulatory factors inside a confined contrary to DXA and bone markers reflect cellular activity space separated from the bone marrow would facilitate of osteoclasts and osteoblasts using the incorporation of a local regulation of the remodeling process without interfer- time marker, namely spaced administration of an agent ence from growth factors secreted by blood cells in the (tetracycline) reflecting ongoing active bone formation. The marrow space. The BRC also creates an environment where study of bone remodeling originated with the classical cells inside the structure are exposed to denuded bone, works of Harold Frost 40 years ago [1] and our ever which may enable direct cellular interactions with integrins expanding understanding of this process is the basis for the and other matrix factors known to regulate osteoclast/ development of highly effective treatments for osteoporo- osteoblast activity. However, the denuded bone surface sis, that we have seen over the last 20 years. inside the BRC also constitutes an ideal environment for the seeding of bone metastases, known to have high affinity for bone matrix. Circulating osteoclast- and osteoblast 2 The bone remodeling cycle precursor cells have been demonstrated in peripheral blood. The dominant pathway regulating osteoclast recruitment is Although macroscopically the skeleton seems to be a static the RANKL/OPG system, while many different factors organ, it is an extremely dynamic tissue at the microscopic level. The ability of bone to sustain the tremendous loads placed on it in everyday life depends on, constant repair of E. F. Eriksen (*) mechanical microdamage that develops both in cancellous Department Of Clinical Endocrinology, Oslo University Hospital, bone—the “spongy” bone present in the vertebrae, pelvis, and Aker, ends (metaphyses) of long bones—and in cortical bone—the Trondheimsveien 235, 0514 Oslo, Norway compact bone present in the shafts (diaphyses) of the long e-mail: [email protected] bones and surrounding cancellous bone as a thin layer in the 220 Rev Endocr Metab Disord (2010) 11:219–227 vertebrae and pelvis. Bone remodeling is based on the [5]. In cortical bone remodeling proceeds in tunnels with concerted action of resorptive and formative cell populations osteoclasts forming “cutting cones” removing damaged in order to replace old bone with new bone and thus secure the bone followed by refilling by osteoblasts in the “closing integrity of the skeleton. This sequence has to be tightly cone” occurring behind the osteoclasts [6]. In normal bone regulated by both local and systemic factors, because the duration of the remodeling cycle in cortical is shorter significant deviations from a neutral balance between resorp- than in cancellous bone with a median of 120 days [6]. The tion and formation would mean severe accelerated bone loss total surface of cancellous bone is completely remodeled or bone gains with possible disastrous consequences in terms over a period of 2 years. of increased fracture risk or compression syndromes. Contrary to remodeling sites in cancellous bone, Bone remodeling takes place in what Frost termed the whichareclosetoredmarrow,knowntocontain Basic Multicellular Unit (BMU), which comprises the osteoprogenitor cells [7], remodeling sites in cortical bone osteoclasts, osteoblasts, and osteocytes within the bone- are distant from red marrow. Therefore, it was assumed remodeling cavity (Fig. 1). In cancellous bone remodeling that the mechanisms of bone remodeling were different in occurs on the surface of trabeculae and lasts about 200 days cancellous versus cortical bone, i.e. that the cells needed in normal bone. The remodeling cycle can be as short as for bone remodeling in cancellous bone traveled directly 100 days in thyrotoxicosis and primary hyperparathyroid- from the red marrow to bone surfaces in cancellous bone, ism and exceed 1,000 days in low turnover states like while cells reached cortical remodeling sites bone via the Myxedema and after bisphosphonate treatment [2]. Remod- vasculature [8]. eling is initiated by osteoclastic resorption, which erodes a resorption lacuna, the depth of which varies between 60 in 2.1 Osteoblast differentiation young individuals and 40 μm in older individuals. The resorption period has a median duration of 30–40 days and Osteoblasts are mesenchymal cells derived from mesoder- is followed by bone formation over a period of 150 days mal and neural crest progenitor cells and their formation (Fig. 1)[3, 4]. In normal bone the result of the remodeling entails differentiation from progenitors into proliferating cycle is complete refilling of the resorption lacuna with new preosteoblasts, bone matrix-producing osteoblasts, and bone. In disease states like osteoporosis, the main defect is eventually into osteocytes or a bone-lining cells. The that the osteoblast is unable to refill the resorption lacuna earliest osteoblastic marker, Runt-related transcription leading to a net loss of bone with each remodeling event factor 2 (Runx2) is necessary for progenitor cell differentiation along the osteoblast lineage [9]. During this sequence of cellular proliferation Runx2 regulates expres- Resorption Formation sion of genes encoding osteocalcin, VEGF, RANKL, Marrow capillary sclerostin, and dentin matrix protein 1 [DMP1] [10]. Cancellous bone Osterix is another transcription factor essential for osteo- 50 µm blast differentiation [11]. A large number of paracrine, autocrine, and endocrine factors affect osteoblast development and maturation like: bone morphogenetic proteins 80 µm (BMPs), growth factors like FGF and IGF, angiogenic factors like endothelin-1, hormones like PTH and prosta- Cortical bone Central vessel glandin agonists, all modulate osteoblast differentiation Haversian system [12]. The action of PTH and BMPs is closely associated with activation of Wnt signalling pathways [13]. 0 100 200 The fully differentiated osteoblast is characterized by Time -days coexpression of alkaline phosphatase and type I collagen, Fig. 1 Schematic representation of Bone Multicellular Units (BMUs) both important for synthesis of bone matrix and in cancellous and cortical bone. Broken lines denote the outer limit of subsequent mineralization thereof [14]. Mature osteo- Bone Remodeling Compartment associated with the resorptive and blasts also produce regulators of matrix mineralization formative sites of the BMU. The mean thickness of the structure in like osteocalcin, osteopontin and ostenectin, RANKL cancellous bone is 50 μm and 80 μm in cortical bone equivalent to a mean Haversian system diameter of 160 μm. The Blood supply for the which is necessary for osteoclast differentiation as well BRCs is provided by capillaries either coming from the marrow space as the receptor for PTH (PTHR1). At the end of their as is the case for cancellous BMUs or from the central vessel of lifespan osteoblasts transform into either osteocytes which Haversian systems in cortical bone. The duration of the remodeling become embedded in the mineralized matrix or lining sequence is somewhat longer in cancellous than in cortical bone. The position of marrow cappillaries is hypothetical, as the exact cells, which cover all surfaces of bone. Specific molecules distribution is poorly elucidated expressed by osteocytes include DMP1, FGF 23 and Rev Endocr Metab Disord (2010) 11:219–227 221 sclerostin, which control bone formation and phosphate reduced estrogen levels favoring increased resorption. In metabolism [15]. many instances [26]. PTH given as daily injections favors bone anabolism, reduces RANKL and increases OPG 2.1.1 Wnts and osteoblast differentiation levels. In cases with chronic elevation of circulating PTH levels as seen in primary hyperparathyroidism the opposite Wnts are secreted glycoproteins crucial for the development pattern is seen with elevated RANKL and reduced OPG and renewal of many tissues, including bone. Wnt levels. A humanized monoclonal antibody against RANKL signalling dominate osteoblast differentiation pathways has been shown to elicit even more pronounced reduction and act via binding to a receptor complex consisting of in osteoclast numbers [27] than bisphosphonates and has LDL receptor-related protein 5 (LRP5) orLRP6 and one of demonstrated excellent reduction of fracture risk in post- ten Frizzled molecules [13]. The so called canonical Wnt menopausal osteoporosis [28]. signaling pathway is active in all cells of the osteoblastic lineage, and involves the stabilization of β-catenin and 2.3 Coupling between resorption and formation regulation of multiple transcription factors [16]. Wnt/β- catenin signaling is also important for mechanotransduc- During normal bone remodeling, the amount of resorbed tion, fracture healing and osteoclast maturation [17–19]. bone is completely replaced in location and amount by new The activation of canonical Wnt-signaling promotes bone. This is secured through tight coupling of bone osteoblast differentiation from mesenchymal progenitors at resorption to bone formation. The mechanisms underlying the expense of adipogenesis, which leads to improved bone the coupling process still remains largely elusive, although strength, while suppression causes bone loss [20]. Canon- the last 15 years has increased our knowledge significantly. ical Wnt signaling in osteoblast differentiation is modulated The dominating hypothesis years ago was that liberation by Runx2 and osterix [21]. of growth factors like IGF 1 and 2 and cytokines embedded Wnt signaling is a prime target for bone active drugs and in bone matrix during bone resorption secured the balance the approaches include inhibition of Wnt antagonist like between resorption and formation during bone remodeling Dkk1, sclerostin, and Sfrp1 with neutralizing antibodies [29]. Later work showing that osteoblastic bone formation and inhibition of glycogen synthase kinase 3 β (GSK3 β), proceeds unperturbed despite lack of bone resorption in the which promotes phosphorylation and degradation of β- presence of defective osteoclasts lacking for example catenin. One of the most promising approaches so far has chloride channels or important factors for ruffled border been inhibition of the osteocyte protein sclerostin, which formation in osteoclasts like c-Src [30] has supplemented exerts tonic inhibition of osteoblast activity [22]. Sclerostin this hypothesis. The important role that osteoclasts play in is the product of the SOST gene, which is mutated and the regulation of bone formation is also corroborated by downregulated in patients with sclerosteosis and van studies on mice lacking c-fos or M-CSF, which display Buchem disease and sclerosteosis [23], which are diseases absence of osteoclasts and defective bone formation [31]. characterized by high bone density. Expression levels of Other system involved in coupling of bone resorption to sclerostin are repressed in response to mechanical loading and bone formation are the transmembrane proteins, ephrinB2, intermittent PTH treatment [24]. Preliminary studies with a which are expressed on osteoblasts and EPH receptor B4 humanized monoclonal antibody against sclerostin have (EphB4), which are expressed on osteoclasts [32]. Also the shown bone anabolism in animals as well as humans [25]. osteoclastic factor sphingosine 1-phosphate (S1P) [33] seems to play a significant role. The interaction of Ephrin 2.2 Osteoclast differentiation and EPH by cell to cell contact promotes osteoblast differentiation and represses osteoclast differentiation. The dominating pathway regulating osteoclast differentia- Secretion of S1P by osteoclast seems to recruit osteoblast tion is the RANKL/RANK/OPG pathway. This pathway is progenitor cells to sites of bone resorption and stimulate based on osteoblasts promoting osteoclast differentiation differentiation of these progenitor cells by stimulating through membrane presentation of RANKL and binding of EphB4 signaling, This causes a shut down of bone this factor to the membrane receptor RANK on mononu- resorption and initiate the formative phase of bone clear osteoclast precursors. Osteoclast differentiation is also remodeling in the so called transition phase. modulated by M-CSF [26]. The promotion of osteoclast differentiation by RANKL is inhibited by the decoy receptor osteoprotegerin (OPG), which is also produced 3 Targeted and non-targeted remodeling by osteoblasts [26] (74). Estrogens increase OPG and decrease RANKL expression in osteblasts, thus favoring Through its constant removal and renewal of damaged bone formation. Postmenopausal bone loss is linked to bone, bone remodeling secures skeletal integrity throughout 222 Rev Endocr Metab Disord (2010) 11:219–227 life. It has become customary to distinguish between Table 1 Osteoblastic and endothelial markers detected on cells lining targeted and non-targeted (stochastic) remodeling. Non- the Bone remodeling Compartment (BRC) vs. vascular endothelial cells as assessed by immuno- and enzyme histochemical staining targeted remodeling denotes regulation remodeling by hormones like PTH, thyroxine, growth hormone and Antigen BRC Vascular endothelium estrogen, but also antiresorptive drugs like bisphospho- – nates may affect non-targeted remodeling. It seems that VEGF + – the main pathway is via modulation of osteoclasts, which Von Willebrand Factor + – then via the coupling between resorption and formation CD 34 + – subsequently affects osteoblast activity. Targeted remod- Alkaline Phosphatase* + – eling secures removal of damaged bone through targeted Osteocalcin + – resorption. Osteocytes are the most abundant cells in Osteonectin + bone, and their death by microdamage has been suggested to IGF 1,2 + – be the major event leading in the initiation of osteoclastic bone TGF β 1,2,3 + – resorption. In normal bone [34]. Resorption lacunae are 3 bFGF + – times more frequent in association with microcracks, indicating OPG + – that remodeling is associated with repair of such microdamage RANKL + – [35]. Damaged osteocytes promote differentiation of osteoclast precursors driven by secretion of M-CSF and RANKL [36]. In cortical bone there is evidence to suggest, that microdamage not only activates new BMUs, but may also EG may direct the movement of existing BMUs as they tunnel through the cortex. It also seems that the degree of damage to the osteocyte network determines osteocyte metabolic responses to loading and influences targeted remodeling [37]. Analysis of the relationship of between mean microcrack length and BMU resorption space density in cortical bone EG Ob indicates that BMUs have an effective area about 40 times CV greater than their actual cross-section, which suggests that osteoclasts in the cutting cone of cortical BMUs are able to OC sense and steer toward microdamage [38]. The relation OC between microdamage and initiation of bone remodeling is further corroborated by the fact that osteoclastic resorption is augmented in old bone [39].

4 The bone remodeling compartment

The work by Hauge et al. [40] demonstrated that the cells in the BMU, even in cancellous bone, were not directly contiguous to the bone marrow, but rather they were covered by a “canopy” of cells forming the outer lining of a specialized vascular structure with the denuded bone surface as the other delineation. The cells of this canopy display all classicial markers of the osteoblastic phenotype (Table 1), and are therefore most probably bone-lining cells, which seem to be connected to bone-lining cells on the upper quiescent bone surface. The structure has been demonstrat- Fig. 2 Different representations of BRC structures in cortical ( panel) and trabecular bone (lower panel). In cortical bone the BRC ed in cortical as well as cortical bone (Fig. 2). In turn, these (outer demarcation by the broken line) is filled with erythrocyte ghosts bone-lining cells on the quiescent bone surface are in (EG) and is located at the closing cone of the Haversian system communication with osteocytes embedded within the bone situated over osteoblasts (OB). A few osteoclasts (OC) are also seen. matrix. Penetrating the canopy of bone-lining cells, and CV denotes the central vessel of the Haversian system. In trabecular bone (lower panel) the outer lining of the BRC is clearly discernible, presumably serving as a conduit for the cells needed in the demarcating a vascular structure on top of osteoblasts (OB). Picture in BMU, are capillaries. upper panel courtesy of Pierre Delmas, Lyon, France Rev Endocr Metab Disord (2010) 11:219–227 223

Angiogenesis is closely associated with bone resorption high concentrations inhibiting bone resorption [51]. Simi- and bone and angiogenic factors like VEGF and endothelin larly, osteoblastic growth and differentiation are inhibited regulate osteoclast and osteoblast activity [41]. In addition by high concentrations of NO, while lower concentrations blood vessels serve as a way of transporting circulating may play a role in regulating normal osteoblast growth and osteoblast [42] and osteoclast precursors [43]tosites in mediating the effects of estrogens on bone formation, undergoing active remodeling. The involvement of vascular mechanotransduction and bone anabolic responses [51]. cells during the initiation of bone resorption is still The dominating isoform of nitrogen oxide synthase (eNOS) unresolved. Is the very first step adhesion of a blood vessel is expressed in osteocytes and lining cells, but not in to bone lining cells at a site where targeted repair is cuboidal osteoblasts [52]. Acidosis and hypoxia generally needed? Conceivably, osteocyte apoptosis and possible increase bone resorption [53–56] and inhibit bone forma- release of osteotropic growth factors and cytokines could tion [57]. As hypoxia may cause acidosis through increased be attractants for blood vessels, which would then subse- anaerobic metabolism, the two factors may act synergisti- quently initiate the formation of a resorptive BRC. But, as cally at the tissue level [56]. Hypoxia and acidosis also outlined above, the framework for signaling within the affect secretion of pro-angiogenic factors like VEGF as osteocyte-lining cell-BRC network could also be a way by outlined below. which remodeling events on bony surfaces are triggered from damage accumulation or changes in mechanical strain within bone. 5 Key functions of the bone remodeling compartment There is increasing evidence for a common lineage and close interaction between vascular endothelial cells and 1. The BRC provides a closed microenvironment permit- bone cells. Endothelial cells drive differentiation of marrow ting tight regulation of bone remodeling. Current stromal cell towards the osteoblastic phenotype [44] concepts regarding local regulation of bone remodeling Endothelin and VEGF are also involved in signaling generally assumes that the local growth factors, between vasculature and bone [45], and VEGF as well as cytokines and even NO come either from cells in the other angiogenic factors are expressed during intramem- marrow space or vascular cells having free access to the branous osteogenesis. Osteoblastic cells, as well as osteo- remodeling site without barriers, or are produced by clasts, possess receptors for VEGF and also produce VEGF osteoclasts and osteoblasts at the remodeling site. The [46]. Expression of VEGF is closely associated with the BRC concept implies that the all factors liberated from early phases of bone modeling and remodeling events [47] the cells or vessels in the marrow space exert their and it induces osteoblast chemotaxis and differentiation regulatory role either through diffusion through the [48] and differentiation. outer layer of the BRC, transport via the bloodstream to Cells may enter the remodeling space either via the interior of the BRC or indirectly via modulation of diapedesis through the lining cell dome covering the BRC cell activity in the outer wall of the BRC. The presence or via the circulation. It is still debatable whether all cells of a specific compartment in which remodeling can involved in remodeling arrive via the circulation, but while proceed without interference from local factors liberat- circulating osteoclast precursors were demonstrated more ed in the marrow space seems to be logical. If the than a decade ago, there is now increasing evidence that access to the marrow space was open, the very high osteoblast lineage cells are also present in the circulation levels of growth factors in the marrow microenviron- strengthening the involvement of circulating precursor cells ment might offset eventual localized regulatory effects in the process [42, 49, 50]. by local growth factors, crucial to osteoclast and While the systemic hormonal regulation of the remodel- osteoblast differentiation and the remodeling process. ing process has to occur via factors arriving at individual 2. The BRC is the structure translating microdamage into remodeling sites via the bloodstream, the way by which targeted remodeling by which mechanosensory signals local regulatory factors exert their action on individual cell from the osteocyte network are translated into changes populations involved is still obscure. Over the last decades, in osteoclast and osteoblast activity on trabecular however, we have increased our knowledge about the surfaces. Lining cells are connected to the osteocyte different growth factors and cytokines involved in local network via gap junctions between lining cells on regulation of bone remodeling tremendously (Fig. 3). Apart quiescent surfaces and osteocyte cannaliculi [58] from growth factors and cytokines, simple molecules like (Fig. 4). Signals from lining cells indicating damage nitrogen oxide (NO), as well as hypoxia and acidosis have or stress could be transmitted to the outer lining cell been shown to exert pronounced effects on bone remodel- layer of the BRC and trigger osteoclast recruitment. By ing balance and activity. NO exerts biphasic effects on analogy with remodeling in cortical bone, which is osteoclast activity with low concentrations potentiating and clearly associated with growth of a blood vessel into 224 Rev Endocr Metab Disord (2010) 11:219–227

MONOCYTES VEGF, FGF, ENDOTHELIN,N Marrow capillary/sinusoids IL1,6,7,11, TNFα O, H+, HYPOXIA E2, PTH, 1,25D,T3

IGF-2, IGF-1, TGFβ,

-- OC OB

IL,M-CSF. C-fos TNF, Osteoblastic Vascular endothelium Lining cells EphrinB2 RANKL EphB4 EphrinB2

Osteoclast precursor EphB4 RANK reseptor

IGFs, S1P Osteoclast Osteoblast Monocyte Osteoclast

Fig. 3 Depiction of some of the main local regulatory factors endothelial cells (vascular endothelial growth factor (VEGF), endo- operating at remodeling sites with osteoclasts (OC) and osteoblasts thelin, nitrogen oxide (NO)) may diffuse to receptors on osteoclasts or (OB). Interleukins (IL), tumor necrosis factors (TNF), transforming osteoblasts. The cellular responses in the BMU are then further growth factors (TGF), colony stimulating factors (CSF), Insulin like modulated by systemic hormones in the circulation (estrogen (E2), growth factors (IGF), fibroblast growth factors (FGF), platelet derived parathyroid hormone (PTH), active vitamin D (1,25D), thyroid growth factors (PDGF), bone morphogenetic proteins (BMP)) are hormone (T3)). Left lower insert depicts in detail osteoblast- formed by both monocytic cells in the marrow space or circulation, as osteoclast interactions inside the BRC and right lower insert depict well as bone cells in the BMU. NFκB- or RANK- ligand (RANKL) an alternativ, still hypohetical, version of that interaction based on and osteoprotegerin (OPG) are formed specifically by osteoblasts. lining cells acting as the osteoblastic component in thata interaction Factors from the marrow space as well as factors liberated by

the remodeling site (5), the presumed ingrowth of a MARROW CAPILLARY capillary into the BRC provides the vascular supply for the cells in the BMU of cancellous bone and might also provide the necessary osteoclasts and, subsequently, the OC osteoblasts that are needed for bone remodeling in both OB cancellous and cortical bone The BRC would also be a site where hormonal modulation (e.g. ERT) of the mechanosensory input could take place [59]. 3. The BRC is the most probable structure at which coupling between osteoclasts and osteoblasts occurs. OSTEOCYTES The RANKL/OPG pathway involves presentation of osteoblastic, membrane bound RANKL to the RANK receptor on osteoclast precursors by cell to cell contact. Fig. 4 Connections between the osteocyte network, lining cells and the Due to the timing and sequence of bone resorption and BRC. All cells in this network are connected with gap junctions, which bone formation, however, resorption and formation are mayprovideapathway(block arrows), by which signals generated deep generally separated in time and space, which makes the within bone may reach the surface and elicit remodeling events by needed cell to cell contact between osteoclast precur- osteoclasts (OC) and osteoblasts (OB) in response to mechanical stimuli. The response may be modulated by factors liberated from the sors and active osteoblasts highly unlikely on a broader vascular endothelium or marrow capillaries/sinusoids and paracrine basis, and even if soluble RANKL played a major role factors (broken arrow) liberated from lining cells may also play a role it had no RANKL on precursor cells within the BRC to Rev Endocr Metab Disord (2010) 11:219–227 225

bind to. A more likely cell, which could present RANKL 6 Conclusion to RANK on osteoclast precursors, would be the lining cell. As demonstrated in animals [60] and in humans Bone remodeling involves tight coupling and regulation of [61], lining cells exhibit positive immunoreactivity for osteoclasts and osteoblasts and is modulated by a wide variety OPG and RANKL, and might therefore be responsible of hormones and osteocyte products secreted in response to for the cell to cell contact with osteoclast precursors. mechanical stimulation and microdamage. Bone remodeling 4. The BRC also obviates the need for a “postal code” proceeds in a specialized vascular entity the “Bone Remodel- system ensuring that resorptive and formative cells ing Compartment” (BRC), which provides the structural basis adhere to areas on the bone surface, where they are for coupling and regulation of cellular activity. Increased needed. Bone surfaces are generally covered by lining knowledge about the interplay between different factors and cells, which would prevent direct contact between bone cells surrounding the BRC will most likely result in even cells and integrins or other adhesion molecules known better treatment options for skeletal disease. to modulate cell activity. The BRC would be the only place where these cells (circulating osteoclasts as well as circulating osteoblast precursors) would be exposed Open Access This article is distributed under the terms of the to these matrix constituents, because the formation of Creative Commons Attribution Noncommercial License which per- the BRC involves detachment of lining cells from the mits any noncommercial use, distribution, and reproduction in any bone surface. medium, provided the original author(s) and source are credited. 5. The BRC may play a crucial role in the spread of bone metastases. It is well established that apart from References entering bone via local ingrowth, tumor cells reach bony surfaces via the circulation. The growth of 1. Frost HM. Tetracycline-based histological analysis of bone metastatic cells in bone is enhanced by the so called – “ ” remodeling. Calcif Tissue Res. 1969;3:211 37. vicious cycle , where PTHrp produced by tumor cells 2. Eriksen EF. Normal and pathological remodeling of human (e.g. breast cancer cells) induces increased local bone trabecular bone: three dimensional reconstruction of the remodel- resorption and subsequent liberation of TGFβ from the ing sequence in normals and in metabolic bone disease. Endocr – bone matrix [62]. The local effects of TGFβ in the Rev. 1986;7:379 408. 3. Eriksen EF, Gundersen HJ, Melsen F, Mosekilde L. 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