J Med Dent Sci 2012； 59： 65－74

Osteoclasts are multinucleated cells of hematopoietic origin which are unique in their ability to resorb bone. Osteoclasts are generated from myeloid progenitors through a progression that involves the fusion of mononuclear precursor cells. The identification of RANK-RANKL signaling as the main signal regulating osteoclast differentiation was a major breakthrough in the bone biology field. In addition remarkable discoveries have been made to broaden the knowledge of the molecular mechanisms of osteoclast formation and differentiation. Despite the vital requirement of osteoclasts in bone modeling and remodeling‚ bone-related conditions l ike osteoporosis, Paget’s disease and rheumatoid arthritis where accelerated bone resorption takes place pose a major socioeconomic burden to the society. Hence, a better understanding of the pathways leading to osteoclast differentiation is vital in successfully managing such diseases. This is an attempt to give a birds-eye-view of the players in osteoclast formation and differentiation in a brief and concise manner.

Key words: osteoclasts, osteoclast differentiation, osteoclastogenesis.

Abbreviations: ADAM: A disintegrin and metalloproteinase. AP: Activator protein. AX-II : Annexin II . Bcl6: B

cell lymphoma 6. Blimp1: B lymphocyte-induced maturation protein-1. BM: Bone marrow. Btk: Brutonʼs tyrosine kinase. CLP: Common lymphoid progenitors. CMP: Common myeloid progenitor. DAP: DNAX-activating protein. DC-STAMP: Dendritic cell-specific transmembrane protein. DcR3: Decoy receptor 3. ECF-L: Eosinophil chemotactic factor-L ERK: Extracellular signal-regulated kinase. FcRγ: Fc receptor commonγ subunit. Flt-3L: Flt-3 ligand. Gab2: Grb-2-associated binder-2. EGF: Epidermal growth factor. GM-CFU: Granulocyte-macrophage colony forming units. GM-CSF: Granulocyte-macrophage colony-stimulating factor. GMP: Granulocyte/macrophage progenitors. HSC: Hematopoietic stem cells. IFN: Interferon. IL: Interleukin. ICAM-1: Intercellular adhesion molecule-1. IKK: IκB kinase. IRF-8: Interferon regulatory factor-8. ITAM: Immunoreceptor tyrosine-based activation motif. JNK: Janus N-terminal kinase. LIF: Leukemia inducible factor. LPS: Lipopolysaccharide. MFR: Macrophage fusion receptor. MAPK: Mitogen-activated protein kinase. M-CSF: Macrophage-colony stimulating factor. MEK: MAPK/ERK kinase. MIP-1α: Macrophage inflammatory protein-1α. MMP: Metalloproteinase. MITF: Microphthalmia transcription factor. MKK: mitogen-activated protein (MAP) kinase kinase. NF-κB: Nuclear factor kappa B. NIK: NF-κB inducing kinase. NFAT: Nuclear factor of activated T cell. OCIL: OC inhibitory ligand. OPG: Osteoprotegerin. OC: Osteoclasts. OCP: Osteoclast precursors. OSCAR: Osteoclast-associated receptor. OSF: Osteoclast-stimulating factor. PLC-γ: Phospholipase Cγ. PI-3K: Phosphatidylinositol 3-kinase. PIR-A: paired immunoglobulin-like receptor-A. RANK: Receptor activator of nuclear factor κB. RANKL: Receptor activator of nuclear factor κB ligand. RANTES: Regulated upon activation normal T-cell expressed and secreted. SCF: Stem cell factor. SDF-1:

Corresponding Author: Neil Alles, BVSc, PhDSection of Pharmacology, Dept. of Bio-Matrix, Graduate School, Tokyo Medical and Dental University.Tel: +81-3-5803-5461/5463　Fax: +81-3-5803-0190E-mail: cnraalles.hpha@tmd.ac.jpReceived April 2, 2012；Accepted June 8, 2012

Review

Osteoclast formation and differentiation: An overview

Niroshani Surangika Soysa1,2), Neil Alles1,3), Kazuhiro Aoki1) and Keiichi Ohya1)

1) Section of Pharmacology, Department of Bio-Matrix, Graduate School, Tokyo Medical and Dental University, Japan2) Division of Pharmacology, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka3) Japan Society for the Promotion of Science (JSPS), Japan

66 J Med Dent SciN. Surangika Soysa et al.

Stromal cell-derived factor-1. STAT: Signal transducers and activators of transcription. TAK1: TGF-β activated kinase1. TAB2: TAK1-binding protein 2. TIZ: TRAF-6-inhibitory zinc finger protein. TNF: Tumor necrosis factor. TNFR: Tumor necrosis factor receptor. TRAF: TNF receptor-associated factor. TRAP: Tartrate resistant acid-phosphatase. TREM: Triggering receptor expressed in myeloid cells. SCF: Stem cell factor. SIRPβ1: signal regulatory protein β1. VEGF: Vascular endothelial growth factor.

1. Introduction

  Bone is a rigid yet dynamic organ where remodeling takes place throughout the life span of an individual without changing much in the shape or the size of bone. The net balance between osteoblastic bone formation and osteoclastic bone resorption maintains the normal bone mass in the adult skeleton. Therefore, any net change in the bone mass reflects a change in these two processes. Osteoclastogenesis and bone remodeling mainly occur within the bone and bone marrow (BM), except in certain pathological bone diseases, such as rheumatoid arthritis, where accelerated osteoclastogenesis takes place at a disease site outside the marrow cavity, thus resulting in abnormal bone resorption.   The understanding of osteoclasts (OC) differentiation and activation comes from the analysis of a family of biologically related tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies that consist of receptor activator of nuclear factor kappa B (NF-κB) (RANK), its ligand RANKL and osteoprotegerin (OPG), which together regulate OC differentiation. Mutations in the genes encoding the RANK and OPG cause bone diseases in humans underscore the crucial role of RANKL/RANK/OPG signaling pathway in regulating bone homeostasis1. This is an attempt to review the players in osteoclastogenesis in a brief and concise manner.

2. Osteoclast precursors (OCP) generation and mobilization

  The first unequivocal evidence for the origin of OC comes from the studies on parabiosis and BM transplants2. OC arise from hematopoietic stem cells (HSC) and pass through a series of differentiation stages resulting in non-dividing, multinucleated bone resorbing cells. OC arise from the common myeloid progenitor (CMP), a bifurcation of monocyte/macrophage lineage (Figure 1). The common lymphoid

progenitors (CLPs) give rise to lymphoid lineage cells such as T lymphocytes, B lymphocytes, and natural killer cells. Interestingly, both CMPs and CLPs can give rise to dendritic cells. The earliest identifiable OCP are the granulocyte-macrophage colony forming units (GM-CFU). Granulocyte/macrophage progenitors (GMP) then further differentiate into mature granulocytes and macrophage/OC/dendritic cell precursors. Therefore, OC can be defined and distinguished from macrophage polykaryons by the macrophage associated antigens, as they do not express like CD11a-c, CD18 and CD143. Expression of CD11b is the best currently known marker of immediate OCP in humans. Early OCP express tartrate resistant acid-phosphatase (TRAP) and metalloproteinase (MMP)-9 and once commit to become OC, these cells express high levels of pp60 c-Src, carbonic anhydrase, and vitronectin receptors. Figure 2 shows the regulation of OC formation from the early non specific differentiation which depends on the PU.1 and microphthalmia transcription factor (MITF) to RANKL dependent cell commitment to OC fate until the development of resorptive capacity by OC.  Mobilization of OCP involves release of cells into the circulation from the BM and homing from the blood stream to peripheral tissues where the immature OCP differentiate into mature OC, which are capable of resorbing bone and express specific osteoclast markers such as TRAP, Cathepsin K, etc. The mobilization of OCP requires a wide range of molecules and include cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-7, IL-3, IL-12, stem cell factor (SCF), and flt-3 ligand (Flt-3L) and chemokines like IL-8, macrophage inflammatory protein-1α (MIP-1α) and stromal cell-derived factor-1 (SDF-1)4. SDF-1/CXCL12, the most powerful stem cell chemoattractant, is a stem cell survival factor and also a regulator of adhesion interactions attaching stem cells to the extracellular matrix or to stromal cells. SDF-1 along with its sole receptor, CXCR4, provides the most powerful retention signal in the BM for hematopoietic stem and progenitor cells. Reduction of the SDF-1 concentration and CXCR4 expression in the BM induced by any factor will lead to cell release to the peripheral blood5. Three CC chemokines, CKβ-8, regulated upon activation normal T-cell expressed and secreted (RANTES), and MIP-1α elicit significant chemotactic responses. MIP-1α binds mainly to the chemokine receptors CCR1 and CCR5, which are highly expressed by OCPs and other cells. RANTES is produced by T lymphocytes, OC, and osteoblasts (OB). Like MIP-1α, RANTES also binds to CCR1 and CCR5.

67Osteoclastogenesis

Figure 1. Hematopoietic and progenitor cell lineages. Hematopoietic stem cells (HSC) divide into two lineage restricted progenitors, the common lymphoid progenitors (CLP) and common myeloid progenitors (CMP). CLP gives rise to T lymphocytes, B lymphocytes, dendritic cells and natural killer cells (NK). The expression FCγRlo/hi, CD34+/-, and CD11+/- have been identified as reliable markers to discriminate between megakaryocyte-erythrocyte progenitors (MEP) and granulocyte/macrophage progenitors (GMP). MEP give rise to mature erythrocytes and megakaryocytes/platelets in the CMP axis while GMP differentiate into various cell types (granulocytes, macrophages, osteoclasts and dendritic cell precursors) depend on the cytokine exposure. Therefore, OC arise from GMP in the presence of M-CSF and RANKL.

Figure 2. Regulation of osteoclast formation and differentiation. The osteoclast arises from the common myeloid progenitors and the initial nonspecific differentiation along the osteoclast pathway is dependent on PU.1 and the MITF of family of transcription factors as well as M-CSF. RANKL commits the OCP to osteoclast fate mediated by molecules such as TRAF-6, Fos and NF-κB (p50/p52). The committed osteoclasts become multinucleated with the fusion of cells under the aegis of molecules such as DC-STAMP and Atp6v0d2. Polarization is required to develop the resorptive capability of mature osteoclasts which require the molecules like c-Src and vitronectin receptor, αvβ3. The osteoclast bone-resorbing activity requires molecules like carbonic anhydrase (CA), H+-ATPase and Cathepsin K.

Resorbing / activated

OC

Polarized OC

OC fusion Pre OC/ Committed OC

Early monocytic cell

Late monocytic cell

GM-CFU Early OCP

PU 1 M-CSF MITF

M-CSF RANKL TRAF-6 Fos p50/52

CA H+-ATPase Cathepsin K

c-Src αvβ3

DC-STAMP Atp6v0d2

Figure 2

PU.1

68 J Med Dent SciN. Surangika Soysa et al.

MIP-1α and RANTES released from activated OBs and other cells may play important roles in their ability to potently stimulate the chemotactic recruitment and RANKL differentiation of pre-OCs and to increase the migration of mature OCs6.

3. Osteoclast differentiation

  The three stages in OC differentiation include early differentiation where HSC proliferate within macrophage lineage, progress into early OCP stage with the expression of calcitonin receptor and TRAP, and a later stage where fusion occurs. The detailed study of osteopetrosis leads to the better understanding of genes that regulate OCP differentiation. Osteopetrosis occurs as a result of deletion of genes such as PU.1, macrophage-colony stimulating factor (M-CSF), c-Fos, RANKL, RANK, TNF receptor-associated factor 6 (TRAF-6), NF-κB, c-Src and so on. PU.1 transcription factor is the earliest identifiable determinant of the OC/macrophage lineage. PU.1 -/- mice have osteopetrosis without either OC or macrophages7. M-CSF, through its receptor, c-fms, provides signals required for their survival and proliferation and the expression of RANK in early OCP. The analysis of the op/op osteopetrotic mice and toothless (tl) mutation in rats, which carry a mutation in the m-csf gene, shows the pivotal role of M-CSF in OC differentiation. Interestingly, the osteopetrotic condition in op/op mice resolves with age as a result of the compensatory cytokines like GM-CSF along with IL-3, vascular endothelial growth factor (VEGF) and Flt-3L 8-11. Similarly, mice lacking csf1r, the gene coding for the M-CSF receptor, c-fms, exhibit osteopetrosis as op/op mice12.

3.1 RANKL-RANK signaling  RANKL is most abundantly expressed as a type Ⅱ transmembrane protein on the cell surface by BM stromal cells, OB and osteocytes,13 and is necessary and sufficient for OC formation from its committed precursors with its receptor RANK. Both RANK and RANKL deficient mice are severely osteopetrotic without OC14,15. OPG is also produced by OB, acts as a decoy receptor for RANKL. The overexpression of OPG prevents OC formation resulting in osteopetrosis in mice, whereas deletion results in osteoporosis 16. The binding of RANKL to RANK recruits TRAF-6 to activate all three mitogen-activated protein kinase (MAPK) pathways, namely ERK (extracellular signal-regulated kinase), JNK (Janus N-terminal kinase), and p38 as well as phosphatidylinositol 3-kinase (PI-3K) and NF-κB

family of transcription factors (Figure 3). TRAF-6 -/- mice are severely osteopetrotic either with abundant, dysfunctional OC17 or without OC18. MAPK-related TGF-β activated kinase1 (TAK1) along with TAK1-binding protein 2 (TAB2), are detected in activated receptor complexes. A dominant-negative form of TAK1 is able to abolish the RANK-induced activation of activator protein-1 (AP-1) and JNK suggesting that TAK1 is important in activation of NF-κB and AP-119,20. Therefore the formation of the TRAF-6-TAB2-TAK1 complex is involved in the RANK signaling pathway and may regulate the formation and function of OC. Gab2 is also identified as a crucial adapter that couples RANK to downstream signaling pathways required for osteoclastogenesis. The loss of Gab2 results in markedly reduced RANKL/RANK-induced OC differentiation, decreased bone resorption, and osteopetrosis, further clarifying its role in OC formation21.  The activation of NF-κB pathway by RANKL is also important, as deletion of both p50 and p52 subunits cause osteopetrosis with the absence of OC 22. The inhibition of the canonical NF-κB pathway by various inhibitors prevents the osteoclastogenesis and related bone resorption23-25. The defective NIK in alternative NF-κB pathway causes mild osteopetrosis and impairs osteoclastogenesis in aly/aly mice26,27. NF-κB is required for the expression of a variety of cytokines including IL-1, TNF-α, IL-6, GM-CSF, RANKL and other growth factors. The stress activated protein kinase, p38, is also involved in mediating signals induced by RANK and is activated via phosphorylation of MAPK kinase (MKK)628. The stimulation of p38 results in the downstream activation of the transcriptional regulator, MITF. MITF is required for the terminal differentiation of OC and mice lacking MITF gene developed osteopetrosis. MITF resides downstream of the M-CSF and RANKL signaling pathways and controls the expression of genes encoding TRAP and Cathepsin K 29 and osteoclast-associated receptor (OSCAR)30.  The ERK1 kinase is also activated by RANKL signaling and is regulated upstream by activation of MAPK/ERK kinase1 (MEK1). Phosphorylation of ERK is increased by treatment with the p38 inhibitors, whereas the ERK inhibitors (PD98059 and U0126) increase phosphorylation of p38, which shows a seesaw-like balance between ERK and p38 phosphorylation suggesting, that osteoclastogenesis is controlled by a balance between the ERK and p38 pathways. Whereas MEK/ERK pathway negatively regulates osteoclastogenesis, the p38 pathway does so positively31. PKC-β, a protein kinase C (PKC) family

69Osteoclastogenesis

enzyme is found to participate in the ERK-activation pathway by M-CSF and RANKL in OCP, thus revealing a functional role in OC differentiation32.  RANKL induces the expression of the AP-1 family of transcription factors. AP-1 refers to a collection of proteins of the Fos (c-Fos, Fra1, Fra 2 and Fos B) and Jun (c-Jun, Jun B and Jun D) families. Fos -/- mice are devoid of OC with normal macrophage differentiation, thus suggesting a block in differentiation at the branch point between monocyte-macrophage and OC 33. Fra-1 compensates for the lack of c-Fos in Fos -/- mice and fully takes over the c-Fos-dependent functions in vivo 34.

3.2 NFATc1   RANKL -RANK i n t e rac t i on ac t i va tes many transcription factors including nuclear factor of activated T cells (NFATc1), a calcineurin and calcium-regulated transcription factor. The presence of NFATc1 in precursor cells prompts them to undergo osteoclastogenesis in the absence of RANKL35. NFATc1 expression is dependent on the TRAF-6-NF-κB and c-Fos pathways, which are activated by RANKL

and Ca2+ signaling as well. NFATc1 regulates OC specific genes, such as calcitonin receptor, TRAP 35, Cathepsin K36, OSCAR37 and β3 integrin38. NFATc1 complexation with AP-1 is necessary for the induction of TRAP, calcitonin receptor and to enhance the auto amplification of NFATc1 itself. NFATc1 complexes with PU.1 and MITF to induce Cathepsin K and OSCAR39. RANKL stimulation induces an elevation of cytosolic Ca2+, thereby activating calcineurin which mediates dephosphorylation and the nuclear translocation of NFATc1. Whereas RANKL is considered the key osteoclastogenic cytokine, NFATc1 seems to be the master of osteoclastogenic transcription factors40.

3.3 Co-stimulatory signals  Certain co-stimulatory receptor signals of RANK namely, triggering receptor expressed in myeloid cells (TREM)-2 and OSCAR are also required for the efficient OC differentiation. For effective osteoclastogenesis, phosphorylation of immunoreceptor tyrosine-based activation motif (ITAM) of the adaptor proteins of TREM-2 and OSCAR (DNAX-activating protein (DAP12) and Fc

Figure 3. Interrelationship of central and costimulatory signal mediated OC differentiation pathways augment by RANK.RANKL-RANK binding recruits adaptor molecules like TRAF-6, which in turn interacts with TAB2/TAK1 to activate the three MAPKs and the IKK complex. This leads to the activation of downstream regulators of OC formation including c-Fos, AP-1 and NFATc1. RANK co-stimulatory receptors containing adaptor proteins FcRγ and DAP12 result in recruitment of Syk kinase and PLC-γ thus calcium signaling which is critical for NFATc1 induction.

70 J Med Dent SciN. Surangika Soysa et al.

receptor common γ subunit (FcRγ), respectively) are necessary. Mice lacking the adaptor proteins DAP12 and the FcRγ are severely osteopetrotic 41,42. BM cells of DAP12-/-FcRγ-/- fail to differentiate into OC with impaired phosphorylation of the Syk showing the functional link between the RANKL and co-receptors (Figure 3). The activation of the tyrosine kinase Syk results in activating the phospholipase Cγ (PLC-γ) which is necessary to increase the intracellular Ca2+ levels42,43 and activating the NFATc1. Activation of tyrosine kinases Tec and Btk by RANK-RANKL signaling also activates the PLC-γ43 (Figure 3).

4. Osteoclast fusion

  Many molecules are implicated in cell-cell fusion inc luding E-cadher in 44, dendr i t ic cel l -speci f ic transmembrane protein (DC-STAMP) 45, integrins, Src family kinases and a disintegrin and metalloproteinase (ADAM) family proteins46. Though ADAM8 and ADAM12 do not mediate fusion by themselves, they do so in vitro. Macrophage fusion receptor (MFR) interaction with CD47 and CD44 mediates the adhesion/fusion to form multinucleated OC 47. Fusion regulatory proteins (FRP-1), which are identical to CD98 molecules, are also involved in the regulation of cell fusion. Antibodies directed against FRP-1 (anti-FRP-1/CD98 antibodies) enhanced homotypic cell aggregation and multinucleated giant cell formation of monocytes with structural and functional properties of OC48. The v-ATPase V0 subunit, d2 (Atp6v0d2), is required to obtain the optimal cell-cell fusion in osteoclastogenesis, even though Atp6v0d2 deficiency does not affect either differentiation of OC or the v-ATPase activity of OC49.

5. Modulation of osteoclastogenesis

5.1 Activation  The pro-inflammatory cytokine, TNF-α, plays a crucial role in OC differentiation. TNF-α acting through TNFR1 augments RANKL induced osteoclastogenesis and promotes in vitro OC formation independently of RANKL through NF-κB and JNK activation, 50 after priming with trace amounts of RANKL51. TNF-α stimulates OB to express RANKL and M-CSF. Furthermore bacterial lipopolysaccharide (LPS) prompts rapid osteoclastogenesis through the TNFR152. Decoy receptor 3 (DcR3), a member of the TNF receptor superfamily, increases osteoclastogenesis through increased synthesis of TNF-α by activating ERK and p38 MAPK but not JNK53. LIGHT, another member

of the TNF superfamily, promotes RANKL-mediated osteoclastogenesis and OC differentiation by a mechanism independent of RANKL54 (Table 1).  IL-1 is a potent stimulator of OC formation and bone resorption, like TNF-α55. IL-1, through its receptor IL-1R, initiates a cascade of signaling converging at the NF-κB pathway. Cytokines like IL-656,57, IL-1158, oncostatin M57, and leukemia inducible factor (LIF) 57 stimulate OC formation by inducing RANKL expression in OB in signal transducers and activators of transcription (STAT)-1 or STAT-3-dependent manner 59. Annexin Ⅱ (AX-Ⅱ), an

Table 1. Modulators of osteoclastogenesisActivating molecules ReferenceTNF-α Kobayashi et al.50, Lam et al.51

LPS Abu-Amer et al.52

DcR3 Yang et al.53

LIGHT Edwards et al.54

IL-1 Yao et al.55

IL-6 Cronstein BN56, Palmqvist et al.57

IL-11 Kudo et al.58

Oncostatin M Palmqvist et al.57

LIF Palmqvist et al.57

Annexin II Li et al.60

OSF Kurihara et al.61

ECF-L Garcia-Palacios et al.62

Activin A Fuller et al.63

TGF-α Takahashi et al.64

EGF Takahashi et al.64

Wnt5a Maeda et al.65

Wnt5b Santiago et al.66

Inhibiting molecules ReferenceOPG Simonet et al.16

ERK Hotokezaka et al.31

Src Shui et al.67

TIZ Shin et al.68

FHL2 Bai et al.69

IFN-γ Takayanagi et al.70

OIP-1 Koide et al.73

OIP-2 Choi et al.74

IL-3 Khapli et al.75

IL-4 Bendixen et al.76

IL-10 Owens et al.77

IL-12/IL-18 Horwood et al.78

OCIL Hu et al.79

IRF-8 Zhao et al.80

MafB Kim et al.81

Bcl6 Miyauchi et al.82

Blimp1 Miyauchi et al.82, Nishikawa et al.83

71Osteoclastogenesis

autocrine/paracrine factor secreted by OC, stimulates OC formation and bone resorption in vitro by inducing BM stromal cells and activates CD4+ T cells to produce GM-CSF. GM-CSF in turn increases OC precursor proliferation and further enhances OC formation. AX-Ⅱ stimulates both the proliferation and differentiation of OCP through production of GM-CSF and RANKL, respectively60. Osteoclast-stimulating factor (OSF) interacts with c-Src to enhance OC differentiation61. Eosinophil chemotactic factor-L (ECF-L), another autocrine factor produce by OC, acts as a regulator of the expression of intercellular adhesion molecule-1 (ICAM-1) on OCP and enhances the effects of RANKL and 1,25-dihydroxyvitamin D3 (1,25-(OH) 2D 3 ) on OC formation by increasing ICAM-1 on OCP. RANKL-RANK engagement induces ECF-L-mediated increase in NF-κB, AP-1 binding activity and JNK kinase activity62. Fuller et al. 63 showed that activin A, a cytokine enriched in bone matrix, which is secreted by both OB and OC, powerfully synergizes with RANKL for induction of OC-like cells from BM precursors depleted of stromal cells. Takahashi and colleagues,64 using a human marrow culture system, have shown that TGF-α and epidermal growth factor (EGF) stimulate bone resorption by increasing the proliferation of OCP. Wnt5a expressed by OB activates the β-catenin-independent non canonical pathway through receptor tyrosine kinase-like orphan receptor (Ror) present on the OCP. Wnt5a-Ror2 seems to enhance RANK expression in OCP by activating JNK and c-Jun, and thereby increase osteoclastogenesis65. Mice deficient in either Wnt5a or Ror2 show impaired osteoclastogenesis65. Wnt5b also plays a role in osteoclastogenesis, through receptor tyrosine kinase Ryk, in addition to Wnt5a66.

5.2 Inhibition  The various levels of RANK signaling can be lead to reduce OC differentiation. The most upstream of the RANKL/RANK/OPG signaling pathway is the action of OPG on RANKL16 (Table 1). In addition, increased in vitro osteoclastogenesis due to inhibitors of MEK1 (U0126 and PD98059) and mammalian target of rapamycin (mTOR) suggests that the RANKL activation of ERK and the Src pathways can also negatively regulate osteoclastogenesis 31,67. TRAF-6-inhibitory zinc finger protein (TIZ) plays a role in OC differentiation potentially by down-regulating the RANK-TRAF-6 signaling68. The expression of positive and negative modulators during the OC differentiation, regulate the strength of the TRAF-6. TIZ is one of the negative regulators of TRAF-6 function. LIM domain-only protein,

FHL2, inhibits OC differentiation and function through binding to TRAF-6, thus inhibiting its association with RANK. Therefore, FHL2-/- OC reach maturation and optimally organize their cytoskeleton earlier and have hyper-resorptive OC69.   There is substantial evidence demonstrating that IFN-γ strongly suppresses osteoclastogenesis in vitro 70. IFN-γ directly blocks OC formation by inducing the degradation of TRAF-6, and thereby inhibition of the RANKL-induced activation of NF-κB and JNK. IFN-γ down regulates the RANKL signaling pathway by antagonizing RANKL-stimulated Cathepsin K 71. Contrary to the aforementioned study, IFN-γ seems to indirectly stimulate OC formation and promote bone resorption by stimulating antigen-dependent T cell activation and T cell secretion of RANKL and TNF-α72. An analysis of the in vivo effects of IFN-γ in 3 mouse models of bone loss - ovariectomy, LPS injection, and inflammation via silencing of TGF-β signaling in T cells, shows that the net effect of IFN-γ in these conditions is that of stimulating bone resorption and bone loss72. Therefore they suggest that the net balance of these 2 opposing forces of IFN-γ is biased toward bone resorption under the conditions of estrogen deficiency, infection, and inflammation.  OC inhibitory peptide 1 (OIP-1) is a negative regulator of OC formation and bone resorption. OIP-1, a glycosyl phosphatidylinositol (GPI)-linked membrane protein, can be cleaved from the cell surface to inhibit OC formation. IFN-γ enhances OIP-1 expression in OCP, GM-CFU and OIP-1 inhibits OC formation through suppression of TRAF-273. In addition OIP-2 inhibits OC formation via its c-terminal fragment through a putative receptor on OCP74. A host of cytokines including IL-375, IL-476 and IL-1077 have negative effects on osteoclastogenesis. IL-3 which shares one of its receptor subunits with IL-4 has similar actions of OC to those of IL-4. IL-10 like IL-4 inhibits OC formation and might be an important matrix protective cytokine during inflammation77. The inhibitory effects of both IL-12 alone and IL-12 combined with IL-18 are T cell dependent through GM-CSF, IFN-γ and yet uncharacterized secreted inhibitory molecules78.  OC inhibitory ligand (OCIL) has the ability to inhibit OC formation and resorption similar to the functions of OPG. OCIL is a type Ⅱ membrane-bound c-lectin expressed by OB, fibroblasts, lymphocytes and hematopoietic cells. OCIL acts in a contact-dependent manner by its transmembrane topology, whereas OPG acts as a secreted inhibitor of OC formation and activity with distant actions from the cells expressing it 79. The expression of several transcription factors,

72 J Med Dent SciN. Surangika Soysa et al.

such as interferon regulatory factor-8 (IRF-8), MafB and B cell lymphoma 6 (Bcl6) in OCP, are down-regulated during the initial phase of OC differentiation, induced by RANKL. Later they act as anti-osteoclastogenic factors by negatively regulating the NFATc1 activity80-82. A recent study shows that RANKL, through the activation of NFATc1, induces the B lymphocyte-induced maturation protein-1 (Blimp1) which acts as a transcriptional repressor of IRF-8, MafB, and Bcl6 genes82,83. The lack of Blimp1 in OC causes the loss of the suppressive effect of Bcl6, thus showing that Blimp1-Bcl6 acts as a negative regulator of osteoclastogenesis82,83.

6. Concluding remarks

  Skeletal disorders like osteoporosis, rheumatoid arthritis are a major health concern and socioeconomic burden to the society. All pathological bone diseases reflect an imbalance between osteoclasts and osteoblasts activity. Osteoclasts, the exclusive bone-resorbing cells play pivotal roles in bone remodeling and pathological bone resorption. Obtaining a better understanding osteoclast regulation is therefore central to the development of new treatments for bone disorders. Recent breakthroughs in understanding the osteoclast biology will therefore allow for the invention of more anti-resorptive therapeutic drugs in the near future.

References1. Crockett JC, Mellis DJ, Scott DI, et al. New knowledge on

critical osteoclast formation and activation pathways from study of rare genetic diseases of osteoclasts: focus on the RANK/RANKL axis. Osteoporos Int 2011; 22: 1-20.

2. Teitelbaum SL. Osteoclasts; culprits in inflammatory osteolysis. Arthritis Res Ther 2006; 8: 201-08.

3. Athanasou NA and Quinn J. Immunophenotypic differences between osteoclasts and macrophage polykaryons: immunohistological dist inction and implications for osteoclast ontogeny and function. J Clin Pathol 1990; 43: 997-1003.

4. Lapidot T and Petit I. Current understanding of stem cell mobilization: the roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells. Exp Hematol 2002; 30: 973-81.

5. Hattori K, Heissig B, Tashiro K, et al. Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells. Blood 2001; 97: 3354-60.

6. Yu X, Huang Y, Collin-Osdoby P, et al. CCR1 chemokines promote the chemotact ic recru i tment , RANKL

development, and motility of osteoclasts and are induced by inflammatory cytokines in osteoblasts. J Bone Miner Res 2004; 19: 2065-77.

7. Tondravi MM, McKercher SR, Anderson K, et al . Osteopetros is in m ice lack ing haematopo iet ic transcription factor PU.1. Nature 1997; 386: 81-4.

8. Yoshida H, Hayashi S, Kunisada T, et al. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 1990; 345: 442-4.

9. Lean JM, Fuller K and Chambers TJ. FLT3 ligand can substitute for macrophage colony-stimulating factor in support of osteoclast differentiation and function. Blood 2001; 98: 2707-13.

10. Myint YY, Miyakawa K, Naito M, et al. Granulocyte/macrophage colony-stimulating factor and interleukin-3 correct osteopetrosis in mice with osteopetrosis mutation. Am J Pathol 1999; 154: 553-66.

11. Niida S, Kaku M, Amano H, et al. Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption. J Exp Med 1999; 190: 293-8.

12. Dai XM, Ryan GR, Hapel AJ, et al. Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Blood 2002; 99: 111-20.

13. Nakashima T and Takayanagi H. New regulation mechanisms of osteoclast differentiation. Ann N Y Acad Sci 2012;1240: E13-8.

14. Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998; 93: 165-76.

15. Li J, Sarosi I, Yan XQ, et al. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci U S A 2000; 97: 1566-71.

16. Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 1997; 89: 309-19.

17. Lomaga MA, Yeh WC, Sarosi I, et al. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev 1999; 13: 1015-24.

18. Naito A, Azuma S, Tanaka S, et al. Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 1999; 4: 353-62.

19. Lee SW, Han SI, Kim HH, et al. TAK1-dependent activation of AP-1 and c-Jun N-terminal kinase by receptor activator of NF-kappaB. J Biochem Mol Biol 2002; 35: 371-6.

20. Mizukami J, Takaesu G, Akatsuka H, et al. Receptor activator of NF-kappaB ligand (RANKL) activates TAK1 mitogen-activated protein kinase kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6. Mol Cell Biol 2002; 22: 992-1000.

21. Wada T, Nakashima T, Oliveira-dos-Santos AJ, et al. The

73Osteoclastogenesis

molecular scaffold Gab2 is a crucial component of RANK signaling and osteoclastogenesis. Nat Med 2005; 11: 394-9.

22. Franzoso G, Carlson L, Xing L, et al. Requirement for NF-kappaB in osteoclast and B-cell development. Genes Dev 1997; 11: 3482-96.

23. Alles N, Soysa NS, Hayashi J, et al. Suppression of NF-kappaB increases bone formation and ameliorates osteopenia in ovariectomized mice. Endocrinology 151: 4626-34.

24. Soysa NS, Alles N, Shimokawa H, et al. Inhibition of the classical NF-kappaB pathway prevents osteoclast bone-resorbing activity. J Bone Miner Metab 2009; 27: 131-9.

25. Jimi E, Aoki K, Saito H, et al. Selective inhibition of NF-kappa B blocks osteoclastogenesis and prevents inflammatory bone destruction in vivo. Nat Med 2004; 10: 617-24.

26. Soysa NS, Alles N, Weih D, et al. The pivotal role of the alternative NF-kappaB pathway in maintenance of basal bone homeostasis and osteoclastogenesis. J Bone Miner Res 2010; 25: 809-18.

27. Soysa NS, Alles N, Takahashi M, et al. Defective nuclear factor-kappa B-inducing kinase in aly/aly mice prevents bone resorpt ion induced by loca l in ject ion of lipopolysaccharide. J Periodontal Res 2011; 46: 280-4.

28. Yamada H, Gursel I , Takeshita F, et al. Effect of suppressive DNA on CpG-induced immune activation. J Immunol 2002; 169: 5590-4.

29. Mansky KC, Sankar U, Han J, et al. Microphthalmia transcription factor is a target of the p38 MAPK pathway in response to receptor activator of NF-kappa B ligand signaling. J Biol Chem 2002; 277: 11077-83.

30. So H, Rho J, Jeong D, et al. Microphthalmia transcription factor and PU.1 synergistically induce the leukocyte receptor osteoc last -assoc iated receptor gene expression. J Biol Chem 2003; 278: 24209-16.

31. Hotokezaka H, Sakai E, Kanaoka K, et al. U0126 and PD98059, specific inhibitors of MEK, accelerate differentiation of RAW264.7 cells into osteoclast-like cells. J Biol Chem 2002; 277: 47366-72.

32. Lee SW, Kwak HB, Chung WJ, et al. Participation of protein kinase C beta in osteoclast differentiation and function. Bone 2003; 32: 217-27.

33. Wagner EF and Eferl R. Fos/AP-1 proteins in bone and the immune system. Immunol Rev 2005; 208: 126-40.

34. Fleischmann A, Hafezi F, Elliott C, et al. Fra-1 replaces c-Fos-dependent functions in mice. Genes Dev 2000; 14: 2695-700.

35. Takayanagi H, Kim S, Koga T, et al. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 2002; 3: 889-901.

36. Matsumoto M, Kogawa M, Wada S, et al. Essential role of p38 mitogen-activated protein kinase in Cathepsin K gene expression during osteoclastogenesis through association of NFATc1 and PU.1. J Biol Chem 2004; 279: 45969-79.

37. Kim K, Kim JH, Lee J, et al. Nuclear factor of activated T

cells c1 induces osteoclast-associated receptor gene expression during tumor necrosis factor-related activation-induced cytokine-mediated osteoclastogenesis. J Biol Chem 2005; 280: 35209-16.

38. Crotti TN, Flannery M, Walsh NC, et al. NFATc1 regulation of the human beta3 integrin promoter in osteoclast differentiation. Gene 2006; 372: 92-102.

39. Asagiri M, Sato K, Usami T, et al. Autoamplification of NFATc1 expression determines its essential role in bone homeostasis. J Exp Med 2005; 202: 1261-9.

40. Takayanagi H. The role of NFAT in osteoclast formation. Ann N Y Acad Sci 2007; 1116: 227-37.

41. Koga T, Inui M, Inoue K, et al. Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis. Nature 2004; 428: 758-63.

42. Mocsai A, Humphrey MB, Van Ziffle JA, et al. The immunomodulatory adapter proteins DAP12 and Fc receptor gamma-chain (FcRgamma) regulate development of functional osteoclasts through the Syk tyrosine kinase. Proc Natl Acad Sci U S A 2004; 101: 6158-63.

43. Shinohara M, Koga T, Okamoto K, et al. Tyrosine kinases Btk and Tec regulate osteoclast differentiation by linking RANK and ITAM signals. Cell 2008; 132: 794-806.

44. Mbalaviele G, Chen H, Boyce BF, et al. The role of cadherin in the generation of multinucleated osteoclasts from mononuclear precursors in murine marrow. J Clin Invest 1995; 95: 2757-65.

45. Yagi M, Miyamoto T, Sawatani Y, et al. DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J Exp Med 2005; 202: 345-51.

46. Verrier S, Hogan A, McKie N, et al. ADAM gene expression and regulation during human osteoclast formation. Bone 2004; 35: 34-46.

47. Vignery A. Osteoclasts and giant cells: macrophage-macrophage fusion mechanism. Int J Exp Pathol 2000; 81: 291-304.

48. Higuchi S, Tabata N, Tajima M, et al. Induction of human osteoclast-like cells by treatment of blood monocytes with anti-fusion regulatory protein-1/CD98 monoclonal antibodies. J Bone Miner Res 1998; 13: 44-9.

49. Lee SH, Rho J, Jeong D, et al. v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation. Nat Med 2006; 12: 1403-9.

50. Kobayashi K, Takahashi N, Jimi E, et al. Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J Exp Med 2000; 191: 275-86.

51. Lam J, Takeshita S, Barker JE, et al. TNF-alpha induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand. J Clin Invest 2000; 106: 1481-8.

52. Abu-Amer Y, Ross FP, Edwards J, et al. Lipopolysaccharide-stimulated osteoclastogenesis is mediated by tumor necrosis factor via its P55 receptor. J Clin Invest 1997; 100: 1557-65.

53. Yang CR, Wang JH, Hsieh SL, et al. Decoy receptor 3 (DcR3) induces osteoclast formation from monocyte/macrophage lineage precursor cells. Cell Death Differ

74 J Med Dent SciN. Surangika Soysa et al.

2004; 11 Suppl 1: S97-107.54. Edwards JR, Sun SG, Locklin R, et al. LIGHT (TNFSF14),

a novel mediator of bone resorption, is elevated in rheumatoid arthritis. Arthritis Rheum 2006; 54: 1451-62.

55. Yao Z, Xing L, Qin C, et al. Osteoclast precursor interaction with bone matrix induces osteoclast formation direct ly by an interleukin-1-mediated autocrine mechanism. J Biol Chem 2008; 283: 9917-24.

56. Cronstein BN. Interleukin-6. A key mediator of systemic and local symptoms in rheumatoid arthritis. Bull NYU Hosp Jt Dis 2007; 65 Suppl 1: S11-5.

57. Palmqvist P, Persson E, Conaway HH, et al. IL-6, leukemia inhibitory factor, and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF-kappa B ligand, osteoprotegerin, and receptor activator of NF-kappa B in mouse calvariae. J Immunol 2002; 169: 3353-62.

58. Kudo O, Sabokbar A, Pocock A, et al. Interleukin-6 and interleukin-11 support human osteoclast formation by a RANKL-independent mechanism. Bone 2003; 32: 1-7.

59. Quinn JM and Gillespie MT. Modulation of osteoclast formation. Biochem Biophys Res Commun 2005; 328: 739-45.

60. Li F, Chung H, Reddy SV, et al. Annexin II stimulates RANKL expression through MAPK. J Bone Miner Res 2005; 20: 1161-7.

61. Kurihara N, Menaa C, Maeda H, et al. Osteoclast-stimulating factor interacts with the spinal muscular atrophy gene product to stimulate osteoclast formation. J Biol Chem 2001; 276: 41035-9.

62. Garcia-Palacios V, Chung HY, Choi SJ, et al. Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing in osteoclast precursors expression of LFA-1 and ICAM-1. Bone 2007; 40: 316-22.

63. Fuller K, Bayley KE and Chambers TJ. Activin A is an essential cofactor for osteoclast induction. Biochem Biophys Res Commun 2000; 268: 2-7.

64. Takahashi N, MacDonald BR, Hon J, et al. Recombinant human transforming growth factor-alpha stimulates the formation of osteoclast-like cells in long-term human marrow cultures. J Clin Invest 1986; 78: 894-8.

65. Maeda K, Kobayashi Y, Udagawa N, et al. Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis. Nat Med 2012; 18: 405-12.

66. Santiago F, Oguma J, Brown AM, et al. Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir. Biochem Biophys Res Commun 2012; 417: 223-30.

67. Shui C, Riggs BL and Khosla S. The immunosuppressant rapamycin, alone or with transforming growth factor-beta, enhances osteoclast differentiation of RAW264.7 monocyte-macrophage cells in the presence of RANK-ligand. Calcif Tissue Int 2002; 71: 437-46.

68. Shin JN, Kim I, Lee JS, et al. A novel zinc finger protein

that inhibits osteoclastogenesis and the function of tumor necrosis factor receptor-associated factor 6. J Biol Chem 2002; 277: 8346-53.

69. Bai S, Kitaura H, Zhao H, et al. FHL2 inhibits the activated osteoclast in a TRAF6-dependent manner. J Clin Invest 2005; 115: 2742-51.

70. Takayanagi H, Ogasawara K, Hida S, et al. T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. Nature 2000; 408: 600-5.

71. Pang M, Martinez AF, Jacobs J, et al. RANK ligand and interferon gamma differentially regulate cathepsin gene expression in pre-osteoclastic cells. Biochem Biophys Res Commun 2005; 328: 756-63.

72. Gao Y, Grassi F, Ryan MR, et al. IFN-gamma stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation. J Clin Invest 2007; 117: 122-32.

73. Koide M, Maeda H, Roccisana JL, et al. Cytokine Regulation and the signaling mechanism of osteoclast inhibitory peptide-1 (OIP-1/hSca) to inhibit osteoclast formation. J Bone Miner Res 2003; 18: 458-65.

74. Choi SJ, Kurihara N, Oba Y, et al. Osteoclast inhibitory peptide 2 inhibits osteoclast formation via its C-terminal fragment. J Bone Miner Res 2001; 16: 1804-11.

75. Khapli SM, Mangashetti LS, Yogesha SD, et al. IL-3 acts directly on osteoclast precursors and irreversibly inhibits receptor activator of NF-kappa B ligand-induced osteoclast differentiation by diverting the cells to macrophage lineage. J Immunol 2003; 171: 142-51.

76. Bendixen AC, Shevde NK, Dienger KM, et al. IL-4 inhibits osteoclast formation through a direct action on osteoclast precursors via peroxisome proliferator-activated receptor gamma 1. Proc Natl Acad Sci U S A 2001; 98: 2443-8.

77. Owens JM, Gallagher AC and Chambers TJ. IL-10 modulates formation of osteoclasts in murine hemopoietic cultures. J Immunol 1996; 157: 936-40.

78. Horwood NJ, Elliott J, Martin TJ, et al. IL-12 alone and in synergy with IL-18 inhibits osteoclast formation in vitro. J Immunol 2001; 166: 4915-21.

79. Hu YS, Zhou H, Myers D, et al. Isolation of a human homolog of osteoclast inhibitory lectin that inhibits the formation and function of osteoclasts. J Bone Miner Res 2004; 19: 89-99.

80. Zhao B, Takami M, Yamada A, et al. Interferon regulatory factor-8 regulates bone metabolism by suppressing osteoclastogenesis. Nat Med 2009; 15: 1066-71.

81. Kim K, Kim JH, Lee J, et al. MafB negatively regulates RANKL-mediated osteoclast differentiation. Blood 2007; 109: 3253-9.

82. Miyauchi Y, Ninomiya K, Miyamoto H, et al. The Blimp1-Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis. J Exp Med 2010; 207: 751-62.

83. Nishikawa K, Nakashima T, Hayashi M, et al. Blimp1-mediated repression of negative regulators is required for osteoclast differentiation. Proc Natl Acad Sci U S A 2010; 107: 3117-22.