Cytokines and transcription factors
OCs form by cytoplasmic, but not nuclear, fusion of precursors derived from myeloid progenitor cells that give rise also to macrophages and dendritic cells.9, 10 The progenitor cells differentiate into OCPs in response to macrophage colony-stimulating factor (M-CSF) and RANKL, but expression of several transcription factors, including PU.1, and a heterodimeric complex of microphthalmia-associated transcription factor (MITF) and transcription factor binding to IGHM enhancer 3 (Tfe3),11 is required earlier in myeloid progenitors to promote their differentiation.12 For example, PU.1 and MITF promote expression of c-fms (the M-CSF receptor),12 and mice deficient in these develop osteopetrosis,13 a condition characterized by radiographically dense long bones in which trabecular bone formed during endochondral ossification is not removed due to failure of OC formation or activity.
M-CSF is an essential osteoclastogenic cytokine expressed by osteoblast lineage cells. It promotes expression of RANK on OCP cell membranes, leaving the RANK+ cells primed to respond to RANKL.14 It mediates OCP proliferation, differentiation, and survival through extracellular signal-regulated kinase (ERK)/growth factor receptor bound protein 2 (Grb-2) and Akt/phosphoinositide 3-kinase (PI3K) signaling.14 M-CSF also signals through a complex comprised of phosphorylated DNAX-activating protein 12 (DAP12) and the nonreceptor tyrosine kinase, spleen tyrosine kinase (Syk),14 which is also activated by costimulatory signaling. Thus, M-CSF has important roles in all aspects of osteoclastic bone resorption.
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–/– mice16 and NF-κBp50/p52 double knockout [dKO] mice17) or 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 signaling20–22 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 TNF receptor-associated factors (TRAFs), particularly TRAFs 1, 2, 3, 5, and 6, which function as adapter proteins to recruit protein kinases.26, 27 Of these, only TRAF6 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/nuclear factor of activated T cells, cytoplasmic 1 (NFATc1); and two others to mediate osteoclast activation (sarcoma [Src]) and mitogen activated protein kinase kinase 6 [MKK6]/p38/MITF) and survival (Src and ERK),26–28 which are discussed in the “Regulation of osteoclast activation” section. TRAF2 positively and TRAF3 negatively regulates OC formation (see below).
NF-κB is a family of transcription factors that includes the signaling proteins, reticuloendotheliosis viral oncogene homolog-A (RelA), p50, Rel B, p52, and c-Rel, which induce expression of genes involved in normal and aberrant immune responses, cell division, differentiation, and movement, and carcinogenesis through canonical and noncanonical pathways.29, 30 Requirement of NF-κB in osteoclastogenesis was discovered unexpectedly when NF-κB p50/p52 dKO mice failed to thrive at weaning as a result of the 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 autoamplification of NFATc1 expression.34 It remains to be determined what the precise role of this transient autoamplification of NFATc1 is and which genes are activated in response to it, but it may be downregulation of constitutively active repressors of RANK signaling35 (see “Constitutively-expressed transcriptional repressors of RANK signaling”).
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 transforming growth factor β (TGFβ)-activated kinase-1 (TAK1), which induce activation of inhibitor of nuclear factor kappa-B kinase subunit gamma (IKKγ; 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 noncanonical pathway occurs more slowly, typically within 3 to 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/cellular inhibitor of apoptosis 1 (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-enhanced or TNF-enhanced OC formation in pathologic states. For example, intratibial injection of murine melanoma cells caused localized osteolysis in wild-type (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. Preclinical 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.
NFATc1 and costimulatory 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 signaling34, 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 costimulatory signaling, which is initiated by ligand binding to immunoglobulin (Ig)-like receptors, such as triggering receptor expressed in myeloid cells-2 (TREM-2) and osteoclast-associated receptor (OSCAR).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, tacrolimus (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 costimulatory 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 rheumatoid arthritis (RA).48 Thus, costimulatory signaling likely enhances OC formation and bone resorption mediated by RANKL through this and other mechanisms in RA.
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 maturation27, 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 450, 51 and production of interleukin (IL)-4 and IL-1035 and Th1 cells express interferon-γ (IFN-γ), 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, they54 and mast cells55 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 cotransfer of a subset of CD11b–/loLy6Chi (lymphocyte antigen 6 complex) OCPs with CD4+ T cells from arthritic mice markedly decreased the severity of arthritis in recombination activating gene 2 (Rag2–/–) recipient mice, suggesting that these subpopulations 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 also been implicated in ovariectomy (Ovx)-induced 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, whereas transgenic mice overexpressing Tregs are protected against Ovx-induced bone loss.15, 59 Some of the discrepancies among these studies may result from 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 cells60 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.
RANKL/RANK mutations cause osteopetrosis in humans
Kindreds with RANKL or RANK deletion mutations have marked osteopetrosis and appear to lack palpable lymph nodes.62, 63 However, obvious immunodeficiencies have not been reported in any of them, suggesting that they may have compensatory mechanisms that maintain normal immune responses. Interestingly, mice deficient in RANK specifically in B cells have normal B cell development.64 This may be important for patients being treated with anti-RANKL drugs, such as denosumab, because these findings in mice suggest that they might not interfere with B cell maturation. To date, no significant increase in infections or other signs of impaired immune responses have been reported in patients in clinical trials of denosumab.19
Activating mutations in the RANK gene are responsible for a number of rare bone diseases, including familial expansile osteolysis, and expansile skeletal hyperphosphatasia,65 in which there is increased localized, rather than generalized OC formation and bone resorption. This focal involvement has similarities to adult Paget's disease, many cases of which have mutations in genes encoding molecules that signal downstream of RANK,66 but it is not known why skeletal involvement is not diffuse in these diseases.