(b) RANKL/RANK and downstream signaling

RANKL is a member of the tumor necrosis factor (TNF) superfamily of proteins and is expressed by osteoblast lineage and other cell types, including T and B lymphocytes (15). In the absence of essential molecules that signal downstream of RANK, such as NF-κB and c-Fos, increased numbers of CD11b+ OCPs accumulate (as in RANK-/- mice (16) and NF-κBp50/p52 double knockout (dKO) mice (17)) or precursors are diverted down the macrophage lineage (as in c-Fos-/- mice) (18). Thus, treatment of patients with anti-RANKL drugs could lead to accumulation of OCPs, which could differentiate into OCs after therapy is discontinued. Such a mechanism could perhaps account for the increase in serum bone resorption markers reported in some clinical trials following cessation of Denosumab (19), but the precise mechanism remains to be determined.

During endochondral ossification, growth plate chondrocytes express RANKL, RANK and osteoprotegerin (OPG) (20). 1,25,(OH)₂D₃, bone morphogenetic protein 2 (BMP2) and Wnt/β-catenin signaling (20-22) regulate RANKL expression by these cells to attract OCPs to growth plates and facilitate rapid removal of newly formed bone, thus preventing osteopetrosis (13). Hypertrophic chondrocytes are the major source of RANKL during endochondral ossification, not osteoblastic cells, as had been thought previously, and osteocytes in bone are the major source of RANKL during bone remodeling and in response to mechanical stress (23-25).

Unlike c-fms, RANK lacks intrinsic kinase activity to phosphorylate and activate downstream signaling molecules. Rank recruits TRAFs, particularly TRAFs 1, 2, 3, 5 and 6, which function as adapter proteins to recruit protein kinases (26, 27). Of these, only TRAF 6 appears to have essential functions in osteoclastic cells (26, 27). RANK/TRAF6 signaling activates four main pathways to induce OC formation: (NF-κB; c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1); c-myc; and calcineurin/NFATc1); and two others to mediate osteoclast activation (Src and MKK6/p38/MITF) and survival (Src and ERK), (26-28), which will be discussed later. TRAF 2 positively and TRAF3 negatively regulates OC formation (see below).

NF-κB is a family of transcription factors, which includes the signaling proteins, RelA, p50, Rel B, p52 and c-Rel, that induce expression of genes involved in normal and aberrant immune responses, cell division, differentiation and movement, and carcinogenesis through canonical and non-canonical pathways (29, 30). Requirement of NF-κB in osteoclastogenesis was discovered unexpectedly when NF-κB p50/p52 dKO mice failed to thrive at weaning due to absence of tooth eruption associated with osteopetrosis because the mice did not form OCs (31, 32). The defect in OC formation in NF-κB dKO OCPs is rescued by either c-Fos or NFATc1 retroviral constructs (33), indicating that they act downstream of NF-κB.

NF-κB appears to cooperate with NFATc2 (which is not required for OC formation) to induce expression of NFATc1, with NF-κB p50 and p65 being recruited to the NFATc1 promoter within 1 hour of treatment of OCPs with RANKL, resulting in transient auto-amplification of NFATc1 expression (34). It remains to be determined what the precise role of this transient auto-amplification of NFATc1 is and which genes are activated in response to it, but it may be down-regulation of constitutively active repressors of RANK signaling (35) (see later).

In the canonical pathway, RANKL binding to RANK leads quickly to formation of a complex on the intracellular cytoplasmic portion of RANK that contains a number of proteins, including TRAF6 and TAK1 (TGFβ-activated kinase-1), which induce activation of IKKκ̃ (also called NF-κB essential modulator (NEMO)). This leads to phosphorylation and subsequent activation of IKKβ, which phosphorylates IκB, an inhibitory NF-κB family protein that holds p65 and p50 heterodimers in an inactive state in the cytoplasm. IκB consequently undergoes rapid degradation by the 26S proteasome resulting in release of p65 and p50 and their translocation to nuclei where they prevent apoptosis of OCPs, thus allowing them to continue differentiating (36, 37). Mice with deletion of IKKβ in OC lineage cells have impaired OC formation and osteopetrosis (36). Interestingly, a constitutively active IKKβ (IKKβ-SS/EE) expressed in OCPs induces their differentiation into OCs in the absence of RANK or RANKL treatment (38), further emphasizing the importance of NF-κB signaling in OC formation.

Activation of the non-canonical pathway occurs more slowly, typically within 3-4 hours of RANKL treatment through the activity of NF-κB-inducing kinase (NIK). This leads to processing of the precursor molecule, p100, to p52, which typically signals in association with RelB. In unstimulated cells, newly synthesized NIK gets bound by TRAF3, leading to NIK proteasomal degradation (39). NF-κB activation by RANKL recruits a TRAF/cIAP E3 ligase complex to RANK leading to cIAP1/2 activation by TRAF2. This targets TRAF3 for ubiquitination and degradation allowing NIK to accumulate and activate IKKα, which phosphorylates p100 and leads to its proteasomal processing to p52 and subsequent nuclear translocation of RelB/p52 heterodimers (29, 36).

NIK, IKKα and p100 do not have a required function for basal OC formation (29, 36). However, they do appear to have regulatory roles in RANKL- or TNF-enhanced OC formation in pathologic states. For example, intra-tibial injection of murine melanoma cells caused localized osteolysis in WT, but not in NIK-/- mice (36). In contrast, TNF-transgenic (TNF-Tg) mice crossed with p100-/- mice developed earlier and more severe joint inflammation and bone erosion than TNF-Tg mice, indicating that p100 limits TNF-induced OC formation and inflammation (40). These studies suggest that strategies to inhibit NIK or increase p100 could reduce bone loss in inflammatory and metastatic bone disease. Pre-clinical studies with a peptide that inhibits NF-κB signaling by binding to NEMO reduced osteoclastogenesis and bone erosion in inflammatory arthritis (41). However, to date there have been no clinical studies reported with this agent.

(c) NFATc1 and Co-Stimulatory Signaling

NFAT transcription factors regulate immune responses as well as cardiovascular, muscle, and neuronal and other cell functions (42). NFATc1 is activated in OCPs by being dephosphorylated by calcineurin, a phosphatase, which is activated by calcium-calmodulin signaling (34, 43) mediated by phospholipase Cγ (PLCγ), which plays a key role by releasing calcium from stores within the cytoplasm (34, 44). NFATc1 is also activated through PLCγ by co-stimulatory signaling, which is initiated by ligand binding to immunoglobulin-like receptors, such as TREM-2 (triggering receptor expressed in myeloid cells-2) and OSCAR (osteoclast-associated receptor) (34). These receptors are expressed on OCPs and they recruit adaptor molecules, such as Fc receptor common γ subunit (FcRγ) and DAP12 leading to phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within these adaptor proteins and activation of downstream signaling. NFATc1 is involved in all aspects of osteoclast formation and activation and seems like a prime target for anti-osteoclast therapy. Indeed the immunosuppressive agents and calcineurin/NFATc1 inhibitors, FK506 and cyclosporine-A, prevent bone loss in inflammatory arthritis because they reduce the inflammation and associated bone resorption (45). However, NFATc1 also positively regulates expression of osterix, a key osteoblastogenic transcription factor, and the net effect of these inhibitors in normal mice is bone loss (45).

The ligands for most co-stimulatory receptors remain unidentified, but OSCAR is activated in OCPs by portions of exposed collagen fibers in resorption lacunae (46). Activation of NFATc1 through RANK and OSCAR in turn induces increased OSCAR expression on OCPs in a positive feedback loop (47). Expression of OSCAR and RANKL is increased in the synovium of joints of patients with RA (48). Thus, co-stimulatory signaling likely enhances OC formation and bone resorption mediated by RANKL through this and other mechanisms in rheumatoid arthritis (RA).

(d) T and B Lymphocytes and Osteoimmunology

The recognition that RANKL is expressed not only by osteoblastic cells, but also by T and B cells and synoviocytes in inflammatory bone diseases and that RANK signaling is involved in immune responses, lymph node formation and B cell maturation (27, 44) spawned the new field of osteoimmunology (49). However, the contributions of T and B cells to the increased osteoclastogenesis in inflammatory bone disease are complex. For example, although T helper (Th) cells express RANKL, T regulatory cells (Tregs) inhibit OC formation through cytotoxic T lymphocyte antigen 4 (50, 51) and production of IL-4 and IL-10 (35) and Th1 cells express INFγ, which inhibits OC formation. Both T cell types are present in inflamed joints of RA patients and the effects of T cells overall appear to be inhibitory (52). Th17 cells are the major subset of RANKL-expressing Th cells in inflamed synovium of RA patients (53). In inflamed synovium, they (54) and mast cells (55) express IL-17, which induces OC formation mainly by increasing RANKL expression by synovial fibroblasts (52), although one study has reported direct induction of OC formation by IL-17 from human monocytes (56). Th17 cells are also inflammatory and cause increased expression of TNF, IL-1 and IL-6 by synovial fibroblasts, which in turn increase their expression of RANKL (52). Interestingly, adoptive co-transfer of a subset of CD11b^–/loLy6C^hi OCPs with CD4+ T cells from arthritic mice markedly decreased the severity of arthritis in Rag2-/-recipient mice, suggesting that these sub-populations of OCPs and T cells can be anti-inflammatory (57). There are also conflicting data about B cell expression of RANKL (43, 51, 58). Further study will be required to explain how these complex positive and negative functions of immune cells lead overall to increased bone resorption in inflammatory bone disease, but they point to additional mechanisms to limit bone resorption.

T cells have been implicated also in ovariectomy (Ovx)-induced the bone loss in mice, but these findings also are somewhat controversial (15). For example, T cell-deficient nude mice appear to be protected from bone loss after Ovx in some, but not all studies (15). Estrogen inhibits differentiation of Th17 cells, but the role of IL-17 in Ovx-induced bone loss is unclear because there are conflicting findings of the effects of Ovx on bone loss in IL-17 receptor-deficient mice (15). Estrogen also increases Treg numbers; but it also regulates T cell production of TNF by inhibiting expression of IL-7, which promotes OC formation. In contrast, estrogen deficiency expands the pool of TNF-producing T cells, while transgenic mice over-expressing Tregs are protected against Ovx-induced bone loss (15, 59). Some of the discrepancies among these studies may be due to differences in the strains of mice used, in study design, or to the positive effects of one set of T cells being negated by those of another set, as appears to occur in RA.

A further twist to the role of T cells in Ovx-induced bone loss is that OCs can function as antigen presenting cells and thus can behave as immune cells to activate T cells (10). For example, they express Fc receptor common γ subunit (FcRγ), major histocompatibility complex (MHC) molecules, CD40, and CD80 (60), just like dendritic cells (60) and express a wide range of cytokines. Therefore, OCs could participate in Ovx-induced T cell proliferation and activation along with or in place of dendritic cells. This positive role could be negated, however, because OCs can also inhibit T cell proliferation and suppress T cell production of TNF and IFNγ (61). These positive and negative effects of immune cells, cytokines, estrogen, and estrogen deficiency emphasize the fact that even in pathologic conditions there are mechanisms to limit excessive tissue destruction.

(a) Integrin- and cytokine-mediated OC attachment to matrix and activation

OCs attach to the bone surface mainly through the vitronectin receptor, αVβ3 integrin (75), in association with kindlin-3 (76), a member of a family of proteins that are recruited to integrin adhesion sites in platelets and leucocytes. Kindlin-3 activates β3 integrin during the early events in OC activation. Humans with kindlin-3 gene mutations and kindlin-3-/- mice are osteopetrotic because their OCs do not form podosomes (77).

Integrin binding to bone matrix proteins, such as osteopontin and bone sialoprotein, activates αVβ3 and recruits Src tyrosine kinase by standard outside-in signaling (78). Src phosphorylates Syk, which recruits the co-stimulatory ITAM protein, Dap12, and Slp76, and these function as an adaptor protein complex for Vav3, a guanine nucleotide exchange protein that activates the small GTPase Rho family members, Cdc42 and Rac (78). αVβ3 and c-fms interact physically, and by inside-out signaling through αVβ3 cause a structural change in αVβ3, which is required for its activation (78). Interestingly, β3 integrin -/- mice have only mild osteopetrosis, perhaps because other integrins can substitute for it in OCs (78). RANK is also physically linked to αVβ3 by Src, forming a complex, which activates Syk, Slp-76, Vav3, and Rac, and in this respect is similar to αVβ3/Src interaction (78). Because the OC is the only cell that forms a ruffled membrane to resorb bone, it is possible that components of this activation step could be targets of future anti-resorptive drugs.

(b) Osteoclast ruffled border formation and bone resorption

Inside sealing zones formed by podosomes, the OC surface area facing the bone is increased significantly by the ruffled border membrane, which is formed by accumulation of cytoplasmic lysosomal secretory vesicle fusion with the cytoplasmic membrane and requires expression of Src (79). Vesicle fusion is promoted by the small GTPase Rab7 and synaptotagmin VII, a calcium-sensing molecule, and by proteins involved in autophagy and extracellular protein secretion, including Atg5, Atg7, Atg4B, and LC3 (80, 81). Accordingly, synaptotagmin VII^−/− osteoclasts have severely defective ruffled border formation (82). H⁺ and Cl^- ions pass through the ruffled border and form HCl to dissolve the mineral component of bone, and proteolytic enzymes, particularly cathepsin K, are secreted to degrade the matrix (75). H⁺ ions are secreted through the V-type H⁺ ATP6i proton pump complex, whereas Cl^- ions pass through a chloride channel encoded by ClCN7.

Src phosphorylates proteins involved in OC activation, including Syk, Pyk2, cortactin, and c-Cbl, which has ubiquitin ligase activity (83). It also mediates RANKL-induced survival signaling in vitro (84), but src-/- OCs have normal survival in vivo (79, 83), perhaps because other Src family members substitute for it. Src is over-expressed in many cancers in which it plays positive roles in proliferation, invasion and metastasis and thus is a therapeutic target in both OCs and tumor cells in metastatic bone disease (83). Small molecular inhibitors of Src have been developed, and of these saracatamib currently is being investigated in metastatic prostate cancer with some promising results as an adjuvant to standard chemotherapy (83). To date no Src inhibitors have been studied in osteoporosis clinical trials.

(c) Osteoclast precursor fusion

High OC nuclear numbers correlate with more aggressive resorption, as is seen in Paget's disease and giant cell tumor of bone. OCP fusion is regulated by dendritic cell-specific transmembrane protein (DC-STAMP), Atp6v0d2, OC-STAMP, and CD9 (85). Atp6v0d2 is a subunit of V-ATPase, a component of the V-type H⁺ ATP6i proton pump complex, which also is involved in OCP-mediated inhibition of osteoblast precursor formation (86), one of a number of unanticipated roles for OCPs and OCs in the regulation of bone formation (9).

NFATc1 and c-Fos play major roles in OCP fusion and activation and in conjunction with MITF and PU.1 variously regulate expression of a number of genes, including, DC-STAMP, OC-STAMP, OSCAR, tartrate-resistant acid phosphatase, cathepsin K, V-ATPase-d2 and the calcitonin receptor (12, 87, 88). Vitamin E (α-tocopherol) also regulates OCP fusion by inducing DC-STAMP expression through activation of mitogen-activated protein kinase 14 and MITF (89). Importantly, administration of α-tocopherol to rats at doses taken by some humans as dietary supplements increased OC numbers in the animals and reduced bone mass, suggesting that excessive Vitamin E consumption could adversely affect bone health (89).

(b) RANKL-mediated inhibition

Some inhibitory mechanisms are actually mediated by RANKL signaling itself within OCPs. For example, activation of c-Fos also induces expression by OCPs of interferon-β (INF-β), which binds to the INF-α/β receptor on OCPs leading to inhibition of OCP differentiation by post-transcriptional reduction in c-Fos protein (101). Mice deficient in INF-β or in a component of the INF-α/β receptor have severe osteopenia due to increased OC formation and activity, emphasizing how important this mechanism is (101). INF-β is used to prevent disease flares in multiple sclerosis with significant efficacy. Although some studies have reported beneficial effects on bone mineral density, patient numbers have been low, and this warrants further study.

(c) Ephrin/Eph and semaphorin/neuropilin/plexin signaling (and osteoclast regulation of osteoblasts)

Ephrins and semaphorins are widely expressed molecules that control communication between cells, including neurons and axons during nervous system development, and endothelial cells and lymphocytes during immune responses and angiogenesis (102-104). These molecules are also expressed in bone and regulate interactions between and functions of osteoclastic and osteoblastic cells (105-107). For example, RANKL-induced c-Fos/NFATc1 signaling increases expression of the ligand, ephrinB2, on the surface of OCPs. Reverse signaling through this ligand when it binds directly to the Eph4 receptor on osteoblastic cells down-regulates c-Fos and NFATc1 expression to limit OC formation; forward signaling through Eph4 stimulates osteoblast precursor differentiation by inhibiting the small GTPase, RhoA (105). Decreased ephrinA1 and EphA1 expression was identified in bones of patients with metastatic of prostate cancer (108) and giant cell tumor of bone (109) by mRNA microarray analysis implicating reduced Ephrin-Eph signaling in osteolytic bone disease.

Semaphorins (Semas) are expressed widely as secreted and membrane-associated proteins; the latter signal through plexins and the former through neuropilins (Nrps). Sema3A is secreted by osteoblasts and OCs, and its binding to Nrp1 on OCPs inhibits RANKL-induced OC formation by inhibiting ITAM and RhoA signaling (110). It also binds to Nrp1 on mesenchymal precursors to stimulate osteoblast and inhibit adipocyte differentiation through canonical Wnt/β-catenin signaling. Accordingly, Sema3A and Nrp1-/- mice have osteoporosis with reduced bone formation. Importantly, treatment of mice with Sema3A inhibited bone resorption and increased bone formation in normal mice and enhanced bone regeneration in a mouse cortical bone defect model (110). Sema4D is membrane-bound and binds to plexin1 on target cells. It is expressed by osteoclasts and inhibits osteoblast differentiation and function by activating RhoA-ROCK, which inhibits insulin-like growth factor-1 signaling (111). Consistent with these findings, sema4D-/- and plexin1-/- mice have high bone mass due to increased bone formation (111). Sema6d is membrane-bound, and by binding to plexin-A1 on OCPs induces OC formation through Trem-2/DAP12/PLCγ-induced NFAT activation as well as podosome formation through Rac-GTP generation in OCs. Accordingly, plexin1-/- mice have marked osteopetrosis, but normal osteoblast function (107). Sema7A is expressed by osteoclasts and osteoblasts and induces monocyte production of free radicals, IL-6, and TNF, suggesting that it may play a role in inflammatory bone disease (107). It promotes OC formation and OCP fusion as well as osteoblast migration in vitro, but full understanding of its role in bone awaits generation of Sem7a-/- mice.

These studies and other studies (9) have revealed complex interactions between osteoclastic and osteoblast cells and that OCs and OCPs have important positive and negative regulatory roles in normal and pathologic bone remodeling that had not been anticipated a decade ago. Further studies will be required to determine exactly where and when these interactions take place in remodeling units to initiate and subsequently stop both resorption and formation, but they suggest that drugs could be developed to enhance or restrict some of these interactions to increase bone mass.

(d) Constitutively-expressed transcriptional repressors of RANK signaling

There are also constitutive mechanisms to inhibit basal OC formation. For example, in the absence of RANKL stimulation, Bcl6 is recruited to the NFATc1, cathepsin K, and DC-STAMP promoters to inhibit osteoclastogenesis. In contrast, RANKL induces removal of Bcl6 from these promoters and its replacement by NFATc1, to mediate osteoclastogenesis (112). Bcl6 is one of a group of constitutively-expressed transcriptional repressors in OCPs, including interferon regulatory factor-8 (IRF-8), Eos, and v-maf musculoaponeurotic fibrosarcoma oncogene family protein B (35). Their expression is down-regulated by RANK signaling. They are also direct targets of B lymphocyte–induced maturation protein 1 (Blimp-1), deletion of which in OCs results in osteopetrosis due to up-regulation of Bcl6 and impaired osteoclastogenesis (112). In contrast, Bcl6-/- mice have increased OC formation and severe osteoporosis. Thus, RANKL/RANK activation of NFATc1 in OCPs not only promotes osteoclastogenesis directly, but it also facilitates it indirectly by repressing expression of negative regulators.