Osteoclast precursors, like other members of the monocyte/macrophage family, are both the source and target of a variety of cytokines. Identification of the key cytokines regulating basal osteoclast formation and function followed the observation that generation of osteoclasts in culture requires contact of their precursors with marrow stromal cells, including osteoblasts 7. Thus, the two essential cytokines promoting osteoclastogenesis are receptor activator of NF-κB ligand (RANKL) 37 and macrophage colony-stimulating factor (M-CSF) 38 (also known as CSF-1), each of which is produced by the marrow stromal cell family.

RANKL is a homotrimeric member of the TNF superfamily 39 and the essential osteoclastogenic cytokine. It is expressed as a transmembrane protein by osteoblasts and their precursors and its production is enhanced by osteoclast-stimulating agents such as parathyroid hormone 40 and TNF-α 4142. In physiological circumstances cell-surface-residing RANKL interacts with its receptor, RANK, on osteoclast progenitors, explaining the requirement for contact between the two cells during osteoclastogenesis. In pathological conditions, such as inflammatory arthritis, RANKL is also expressed by activated T lymphocytes and in this circumstance is cleaved from the membrane and functions as a soluble ligand. In fact, T cell-produced RANKL is a major contributor to inflammation-mediated periarticular bone loss 43.

The unique osteoclastogenic properties of RANKL are due to specific structural features of loop components of its external domain, absent from other members of the TNF superfamily, that enable it to recognize its receptor 39. RANK activation, in turn, recruits a number of TNF-receptor-associated proteins (TRAFs). However, it is TRAF6 that endows RANK with its unique osteoclastogenic potential. Although TRAF6 also associates with CD40 and the IL-1 and Toll-like receptors, it does not do so as abundantly as with RANK, probably accounting for at least a significant component of their lack of osteoclastogenic capacity 4445.

Osteoclast recruitment and function are also regulated by the LIM domain-only protein, FHL2, which binds TRAF6 and thus inhibits its association with RANK 46. FHL2 is not detectable in naive osteoclasts in vivo but appears under the influence of RANKL or in animals with inflammatory arthritis. Establishing functional relevance, mice lacking FHL2 have hyper-resorptive osteoclasts and enhanced bone loss stimulated by RANKL and inflammatory arthritis. The accelerated resorption in this circumstance is due to aggressive organization of the osteoclast cytoskeleton, reflecting the capacity of RANKL to activate the mature resorptive cell in addition to promoting osteoclast differentiation 374748.

The osteoclast-activating properties of RANKL are mediated via a complex composed of its receptor, TRAF6 and c-Src, which the cytokine specifically recruits to lipid rafts. Reflecting the cytoskeletal impact of c-Src, this event involves the organization of fibrillar actin and is mediated via the phosphoinositide 3-kinase (PI-3K)/Akt pathway, which also exerts an anti-apoptotic effect on the cells 49.

The discovery of the pivotal role of RANKL in the osteoclastogenic process actually followed on that of the secreted protein, osteoprotegerin (OPG) 50. OPG, like RANKL, is synthesized by osteoblasts and their precursors and is also a member of the TNF superfamily 51. It recognizes RANKL and thus functions as a decoy receptor, competing with RANK for its ligand. As would be predicted, OPG overexpression results in the arrest of osteoclastogenesis and hence leads to osteopetrosis 50. Alternatively, deletion of the OPG gene, Tnfrsf11b, results in severe osteoporosis due to increased osteoclast number and activity 52. Importantly, many of the same resorptive agents that enhance RANKL secretion suppress OPG production, and the ratio of the two molecules dictates the rate of bone loss in a variety of pathological conditions 53.

Activation of the RANK/TRAF6 composite induces a series of intracellular signaling pathways, each of which participates in the osteoclast phenotype. Activation of calcinurin by RANKL-enhanced intracellular calcium is among the most important of these events. Activated calcinurin dephosphorylates nuclear factor of activated T cells 1 (NFAT1), which translocates to the nucleus where, in association with c-Fos and c-Jun, it induces NFAT2 gene expression 54. NFAT2, also in the context of the same AP-1 proteins, has the central role in the transactivation of osteoclastic genes such as tartrate-resistant acid phosphatase, the β₃ integrin subunit and the calcitonin receptor 55. Thus, whereas RANKL is the key osteoclastogenic cytokine, NFAT2 seems to be a key osteoclastogenic transcription factor.

The NF-κB family of transcription factors is also downstream of RANKL and central to osteoclast differentiation. In fact, deletion of the p50 and p52 NF-κB subunits, in concert, completely arrests osteoclastogenesis, resulting in severe osteopetrosis 56. This realization prompted exploration of the NF-κB signaling pathway in the context of the osteoclast, and several intermediary signaling molecules have been identified as crucial to the event.

NF-κB activation occurs via both classical (canonical) and alternative signaling pathways. In both circumstances the IκB kinase (IKK) complex initiates the activation of NF-κB. This complex consists of three subunits, namely IKKα and β, which are catalytic, and IKKγ, which is regulatory. There is little question that IKKγ (also known as NEMO) is essential to the osteoclastogenic process because inhibition of its association with the α and β subunits, by cell-permeable peptides, arrests RANKL-induced osteoclastogenesis and prevents both the inflammatory and bone-destructive components of antigen-induced 57 and serum-transfer arthritis 58.

IKKβ activates the classical pathway by phosphorylating the cytosolic NF-κB binding proteins, IκBs, thereby targeting them for ubiquitin-mediated degradation. Most NF-κB subunits, particularly p65 and p50, are thus liberated and free to translocate to the nucleus and to function as transcriptional regulators. Importantly, the direct administration of non-degradable IκB peptides to mice prevents the development of inflammatory arthritis and its attendant bone destruction 5960.

The IKKβ-activated classical pathway generates osteoclasts in response to RANKL and participates in the bone-destructive components of inflammatory arthritis by promoting the differentiation of osteoclasts and prolonging their lifespan 6162. There is, however, disagreement about the role of IKKα in basal and pathological osteoclastogenesis. IKKα modulates the alternative pathway leading to the generation of p52 NF-κB subunits 63. On the one hand, mice lacking NF-κB-inducing kinase (NIK), which activates IKKα but not IKKβ, are resistant to RANKL-induced osteoclastogenesis and the bone destruction attending a variety of forms of inflammatory arthritis 64. The fact that IKKα^-/- mice exhibit defective osteoclast formation in vivo is in keeping with these NIK-based observations 65. On the other hand, mice bearing an IKKα-inactivating mutation mirror wild-type animals as regards lipopolysaccharide-induced osteoclastogenesis and periarticular osteolysis 61. Although specifics remain to be resolved, the NF-κB signaling pathway is clearly central to physiological and pathological bone resorption and its various components represent potential therapeutic targets.

M-CSF promotes the survival, proliferation and maturation of monocyte/macrophage precursors. It recognizes only one receptor, the tyrosine kinase c-Fms. The central role of the cytokine and its receptor in osteoclastogenesis is established by the fact that op/op mice, with a loss of function mutation in the Csf1 gene 38, and those deleted of c-Fms 66, lack osteoclasts and develop osteopetrosis. Interestingly, the osteopetrotic lesion of op/op mice resolves with age, reflecting a progressively increasing expression of granulocyte/macrophage colony-stimulating factor 67 and vascular endothelial growth factor 68, which compensate for the absence of M-CSF.

Like RANKL, M-CSF production by osteoblasts and their precursors, or by T cells, is stimulated by a variety of osteoclastogenic molecules, often with pathological consequences. For example, c-Fms activation participates in the bone loss attending inflammatory arthritis 69. In this circumstance, inflammation-enhanced IL-1 and TNF-α stimulate the release of IL-7 from stromal cells, which in turn prompts T cells to produce M-CSF. Similarly, increased levels of parathyroid hormone promote the release of M-CSF from osteoblasts and stromal cells in the bone microenvironment 70. An analogous scenario may hold for estrogen deprivation, perhaps participating in the pathogenesis of post-menopausal osteoporosis 71.

Activation of c-Fms involves its dimerization and auto-phosphorylation on specific tyrosine residues. The occupied receptor transmits a variety of signals affecting a broad array of events within the osteoclast and its precursor. For example, M-CSF-induced osteoclast precursor proliferation is mediated by both Erk1/2 and PI-3K/Akt. The latter also prolongs longevity of the mature cell 28. Prolonged Erk activation stimulates osteoclast differentiation via the induction of c-Fos and, probably, NFAT2 28. M-CSF also regulates macrophage and osteoclast migration via cytoskeletal organization mediated by PI-3K and c-Src 7273. The guanine nucleotide exchange factor Vav is phosphorated in response to M-CSF, leading to Rac-stimulated motility 3174.

SHIP1 is a 5' lipid phosphatase that dephosphorylates phosphatidylinositol 3,4,5-trisphosphate and thus inactivates Akt. SHIP1-deficient osteoclasts and their precursors are also hypersensitive to M-CSF 75. A lack of SHIP1 therefore accelerates macrophage proliferation and dampens osteoclast apoptosis. These distinct effects of SHIP1 deletion on osteoclasts and their precursors result in increased numbers of enlarged, hypernucleated cells that aggressively resorb bone and produce an osteoporotic phenotype.

As demonstrated by the above, both M-CSF and the α_vβ₃ integrin activate several of the same signaling pathways in the osteoclast. In fact, they collaborate in osteoclast regulation. For example, the capacity of M-CSF to organize the cell's cytoskeleton depends on α_vβ₃-mediated matrix adhesion 76. Furthermore, the retarded differentiation and cytoskeletal function of β₃^-/- osteoclasts are rescued by a high dose of M-CSF 28. These findings reflect at least one common signaling pathway emanating from the integrin and c-Fms, involving prolonged activation of Erk leading to increased c-Fos expression. The essential role of c-Fos in α_vβ₃-mediated osteoclast cytoskeletal organization is confirmed by rescue of β₃^-/- osteoclasts by overexpression of the AP1 transcription factor 28. M-CSF and α_vβ₃ also share Rac as a common downstream target in osteoclast cytoskeletal organization, an event mediated in both circumstances by activation of Vav3 31.

Rheumatoid arthritis is a complicated condition because a host of cytokines, produced by a variety of cells, contributes to its pathogenesis. Although RANKL and IL-1 are important participants in the development of focal bone erosions that result in joint collapse, TNF-α is the principal and rate-limiting culprit in that its blockade dampens both the inflammatory and osteoclastogenic components of the disease.

TNF-α binds to two distinct receptors, each of which is expressed by osteoclast precursors. However, the osteoclastogenic properties of TNF-α are mediated via its p55 receptor (p55r). Marrow derived from mice expressing only this receptor generate substantially more osteoclasts in response to the cytokine than do the wild type, whereas those bearing only the other TNF receptor, p75r, produce fewer 77. In keeping with this observation, soluble TNF-α, which preferentially activates p55r, has potent osteoclastogenic properties whereas those of its membrane-residing precursor, which recognizes p75r, are negligible. Similarly, whereas lipopolysaccharide seems to mediate osteoclast formation via its Toll-like receptors, it also stimulates the process via p55r 34. TNF-α and RANKL are synergistic, and minimal levels of one markedly enhance the osteoclastogenic capacity of the other 41. Alternatively, TNF-α recruits osteoclasts when precursors are exposed to, or primed by, permissive (that is, constitutive) levels of RANKL 41. This observation in vitro is in keeping with the fact that OPG-treated mice fail to generate an osteoclastogenic response when subjected to inflammatory arthritis 43. Thus, in the presence of M-CSF, RANKL – but not TNF-α – is necessary and sufficient to generate osteoclasts.

Many of the signaling pathways induced by p55 TNF receptor mirror those emanating from activated RANK, calling into question the reason that TNF-α on its own is incapable of promoting osteoclast differentiation. The most compelling evidence in this regard relates to the association of TRAF6 with the RANKL but not the TNF receptor.

TNF-α is a promiscuous cytokine, produced and recognized by a host of cells that participate in inflammatory osteolysis. Marrow stromal cells and macrophages are particular targets of TNF-α in this condition, but the greater contribution to osteoclast recruitment is made by the stromal cells 42. In the presence of relatively mild inflammatory conditions, such as particle-induced implant loosening, TNF-α exerts its effect by stimulating stromal-cell production of cytokines, including RANKL, IL-1 and M-CSF, which in turn target macrophages to promote osteoclast differentiation. As the magnitude of inflammation and TNF-α production increases, substantial osteoclastogenesis is achieved by direct targeting of the cytokine to the osteoclast precursor even in the absence of TNF-α-responsive stromal cells.

M-CSF produced by stromal cells is particularly important in the pathogenesis of TNF-α-induced osteolysis. M-CSF stimulates RANK expression by osteoclast precursors and mediates the capacity of TNF-α to increase the number of these mononuclear cells. Most importantly, M-CSF inhibition selectively and completely arrests the profound osteoclastogenesis attending this condition or after TNF-α administration.

TNF-α enjoys an intimate relationship with IL-1 in pathological bone loss, including that attending loss of ovarian function 7879. In this circumstance, decreased estrogen levels promote interferon-γ expression by T cells. The interferon enhances MHC class II expression by antigen-presenting cells, which in turn promotes T cell proliferation and their production of TNF-α and IL-1. These two cytokines stimulate RANKL expression by stromal cells, thereby increasing osteoclast number, which characterizes the accelerated bone loss of post-menopausal osteoporosis.

IL-1 mediates a substantial portion, but not all, of TNF-α-induced osteoclast recruitment in inflammatory osteoclastogenesis 80 (Fig. 2). Under the aegis of the p38 MAP kinase, IL-1 stimulates RANKL production by marrow stromal cells and, in the context of constitutive RANKL, directly promotes osteoclast precursor differentiation. Like TNF-α, IL-1 on its own is incapable of osteoclast recruitment despite a single TRAF6-binding site on the IL-1 receptor-associated kinase, IRAK. Attesting to their interdependence, blockade of either TNF-α or IL-1 does not completely arrest the periarticular damage of inflammatory arthritis, whereas inhibition of the two cytokines in combination is substantially more effective 81. Thus, TNF-α signaling through p38 MAP kinase induces stromal-cell expression of IL-1, which upregulates its own receptor. Occupancy of the now abundant IL-1 receptor similarly activates p38, which promotes RANKL production. In macrophages, TNF-α enhances RANK expression and the synthesis of IL-1 whose functional receptor is in turn upregulated by the same three cytokines. Thus, the interdependence of TNF-α, RANKL and IL-1 in the generation of osteoclasts lends credence to the observation that combined blockade is most effective in preventing pathological bone loss.