RANK, like other members of the TNFR family, lacks the capability for intrinsic enzymatic activity through its intracellular domain, and relies principally on TNFR associated factor (TRAF) for signal transduction. The cytoplasmic domain of RANK directly interacts with five of the six TRAF proteins, with different binding sites, that of TRAF6 being more proximal to the membrane [21,22](Figure 1-B). Three RANK TRAF-binding motifs have been shown to be involved in the osteoclast formation and activation, as in the RANK-induced activation of NF-κB and MAP kinases JNK, ERK and p38 [23]. In addition, TRAF6-specific binding sites have also been identified, and TRAF6 signaling appears crucial in osteoclast signaling [24-26]. The binding of RANKL to its receptor RANK results in the recruitment of TRAF6; activated TRAF6 may then stimulate NF-κB activity by activation of the IκB kinase (IKK) complex, either through aPKC or TAK1-dependent phosphorylation, a process that requires NEMO (IKKγ) ubiquitination for optimal activation [27](Figure 2). The p62 scaffolding protein is one of the functional links reported between RANKL and TRAF6-mediated NF-κB activation [1]. Once bound to TRAF6, the p62-TRAF6 complex interacts with atypical protein kinase C (aPKC) proteins resulting in the formation of a multimeric protein complex that regulates NF-κB activation via the phosphorylation of IκB kinase β (IKKβ) [28-30]. The p62 complex also binds the scaffolding receptor interacting protein (RIP) and phosphoinositide-dependent kinase 1 (PDK1), recruiting aPKCs to TNF-α signaling complexes [31,32].

TRAF6 also forms complexes with TGFβ-activated kinase 1 (TAK1) and adaptor proteins TAB1 and TAB2 [33]. When TAK1 is activated, it in turn phosphorylates NF-κB-inducing kinase (NIK), which activates the IKK complex, leading to NF-κB pathway activation. As for the MAP kinases, TAK1 also activates the JNK pathway, while TAB1 recruits and binds p38 to the TRAF6 complex, leading to activation of its pathway [34,35]. In addition, RANKL-induced TRAF6 activates the Akt/PKB pathway through a signaling complex that involves c-Src [36]. All these signaling molecules contribute to osteoclast differentiation, survival and activity [9,20]. As a scaffold, p62 plays a major role in RANK-induced signaling, through the recruitment and formation of signaling complexes, and its ability to bind and activate TRAF6 as described below. Confirming the major role of p62 in the RANK/RANKL signaling pathway, genetic inactivation of SQSTM1 leads to the inhibition of IKK and NF-κB activation induced by RANKL, as well as NFATc1 synthesis, and to impaired osteoclastogenesis in mice [28].

In a murine model, RANKL stimulation has been reported to induce the formation of a ternary complex between TRAF6, p62 and atypical PKCs (aPKCs), identifying p62 as an important mediator during osteoclastogenesis [28]. Consistent with this data, it has also been shown in human osteoclast cultures that following RANKL stimulation, PKCζ, P-PKCζ/λ and P-PDK1 as well were associated to p62 [37]. This is consistent with the knowledge that PKCζ, like other aPKCs, is a substrate of PDK1, indicating a role for PDK1 in p62-aPKC signaling, and perhaps other TRAF6-p62 signaling pathways. Indeed, PDK1, a serine/threonine kinase, is a recognized master protein kinase in the regulation of many cell-signaling pathways, known to be constitutively active, and can be further activated by tyrosine phosphorylation (Tyr⁹ and Tyr^373/376). Many proteins involved in the PI-3K signaling pathway, like PI-3K, Akt, and the phosphatase and tensin homolog on chromosome 10 (PTEN) are mediated by PDK1 [38]. PDK1 has also been found to phosphorylate and activate other members of the cAMP-dependent, cGMP-dependent, and protein kinase C kinase families of proteins including both novel and atypical PKCs, p70S6K, and others [39]. However, its exact role in osteoclast p62 signaling remains to be elucidated.

The IKK complex is cardinal to the activation of NF-κB, and is composed of three subunits, one of which is regulatory, called IKKγ or NEMO, and two that are catalytic, IKKα and IKKβ. Notably, in non-osteoclastic cells, p62 has been shown to regulate IKK activation through its ability to bind the atypical PKCs like PKCζ and PKCλ, who favor phosphorylation of IKKβ [29,40]. In addition, in PC12 cells, IKKβ has been shown to be recruited to the multiprotein complex including p62, TRAF6 and aPKC [41].

The protein p62 appears to be a molecular adaptor associating not only TRAF6 but also the de-ubiquitinase CYLD [42]. p62 interactions with TRAF6 stimulate TRAF6 K63-linked autoubiquitination and E3 ligase activity, and regulate the synthesis of K63 chains on target substrates. In addition to the UBA domain, the PB1 domain of p62 is needed for TRAF6 polyubiquitination [1]. Ubiquitination of TRAF6 is an important mechanism mediating its signaling functions [43,44]. However, rather than an absolute requirement for TRAF6 E3 ligase activity in mediating downstream signaling, K63-linked ubiquitination of TRAF6 should be viewed as a marker of activation [27]. In addition to its well established role in the activation of TRAF6, p62 may also regulate a de-ubiquitinating enzyme (DUB) with a specificity for Lys⁶³ (K63) chains (CYLD), that interacts with TRAF6, and reverses the processing of protein ubiquitination. The decrease in the activity of CYLD leads to the accumulation of Lys⁶³ (K63)-ubiquitinated substrates [42]. p62 interacts with CYLD and promotes the binding of CYLD to TRAF6, and this molecular interplay requires the C-terminal domain of p62 [45]. CYLD negatively regulates NF-κB activity by reducing TRAF6 autoubiquitination [46,47]. CYLD negatively regulates the activation of IKK and JNK, and its expression is markedly upregulated under conditions of RANKL-induced osteoclastogenesis [45]. Thus CYLD appears to be a crucial down-regulator of RANK signaling in osteoclasts. Accordingly, CYLD-deficient mice display severe osteoporosis linked to aberrant osteoclast differentiation and have larger and more numerous osteoclasts, which are hypersensitive to RANKL [45].

RANKL specifically and strongly induces the nuclear factor of activated T cells (NFATc1 or NFAT2), a master regulator of osteoclast differentiation [48]. This induction is dependent on TRAF6/NF-κB and c-Fos pathways, as well as calcium signaling. NFATc1 also specifically autoregulates its own promoter particularly during RANKL-induced osteoclastogenesis [9]. Naturally, TRAF6 activity is affected by p62 interaction, affording p62 some measure of indirect control over NFATc1 signaling. However, kinases may also contribute to the nuclear shuttling of this factor, as PKCζ has been shown to interact with NFATc1, and may modulate NFAT-mediated transcription by increasing the activity of its N-terminal transactivation domain [49]. In addition, RANKL also induces MEK/ERK pathway which contributes to osteoclast differentiation and survival [50]. The p62 UBA domain plays an important role in the activation of NF-κB, NFAT and in ERK phosphorylation [51].

The signal for targeting proteins for degradation by either the autophagy or proteasome pathways is ubiquitination, both processes in which p62 plays a major role through binding ubiquitinated protein and trafficking as well. p62 may be involved in the formation of multimeric protein complexes as a result of the interactions of its PB1 domain with other proteins, and it may also play a role in protein turnover as a result of its UBA domain at its C-terminus, which binds non-covalently to polyubiquitin chains, and finally through its association with the proteasome (26S and S5a subunits) via its N-terminus domain [19]. In a well-characterized neuronal model, p62 has been shown to direct the addressing of protein complexes containing the neurotrophin receptors, TRAF6, and PKCζ, making it an essential player in signaling protein degradation and recycling [52,53].

Autophagy takes place in all cells, in order to maintain cell homeostasis or in response to stress or starvation, by eliminating damaged components, and providing cells with energy and nutrient resourcing [54,55]. p62 has been identified as an LC3-interacting protein implicated in autophagy [56-58]. Studies have shown that p62, along with ubiquitinated proteins, is transported into autophagosomes, suggesting that p62 is a receptor for these proteins that directs them to lysosomes [56,58]. This is made possible by its LRS (LC3 recognition sequence) located between the zinc finger and UBA domains of p62, where residues interact with the N-terminus and ubiquitin domain of LC3 [59,60]. Interestingly, knockout of p62 does not appear to markedly affect levels of ubiquitinated proteins in the cell, possibly due to compensatory action by Nbr1 (neighbor of BRCA1 gene 1), which interacts with LC3 in a similar manner [57,61]. Loss of function of Nbr1 leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis [62]. Autophagy has been involved in hypoxia-induced osteoclast differentiation [63], and p62 may play a role in the starvation-induced autophagy in human osteoclasts [4].

The consequences of autophagy mainly favor cell survival, although a complex crosstalk exists between autophagy and apoptosis pathways [55]. While p62 has been involved in the selective autophagic degradation of many proteins, p62 is also involved in several apoptotic and survival pathways. RANKL stimulation contributes to osteoclast survival through p62-driven activation of NF-κB and MEK/ERK pathways [51]. Conversely, p62 also interacts with caspase-8, and is crucial for efficient caspase-8 activation by promoting aggregation of cullin-3 mediated-polyubiquitination [64]. On the other hand, p62 may be cleaved by caspase-6 and -8 in response to death receptor activation [65], and caspase-8, as well as p62, may be degraded by autophagy [66]. Thus, interrelationships exist between autophagy that affect the efficiency of apoptosis, and apoptosis that affects p62-dependent autophagy [55].

The significance of the p62^P392L mutation has been studied in a series of in-vitro experiments, using osteoclast precursors derived from the peripheral blood of PDB patients carrying the p62^P392L gene, and from bone marrow cells or cord blood monocytes (CBM) of normal subjects transfected with the wild-type p62 (p62^wt) or the p62^P392L gene. These osteoclast precursors are hyper-responsive to osteoclastogenic factors, such as RANKL and TNFα, and the p62^P392L-transfected cells have an enhanced ability to resorb bone [68]. Transgenic mice expressing the p62^P394L gene (p62^P392L equivalent in human) develop PDB-like bone lesions [69], as transgenic mice coexpressing the measles virus nucleocapsid (MVNP) under the TRAP promoter and knocked-in p62^P394L [70]. Previous findings have revealed that in human osteoclasts, even prior to RANKL stimulation, p62 associates with both activated PDK1 and PKCζ or activated PKCζ/λ in PDB osteoclasts, in osteoclasts from healthy donors harboring the p62^P392L gene, and in p62^P392L transfected osteoclasts [37]. Although both wild-type and mutated p62 over-expression favors the formation of activated PKCζ/λ-p62 complexes, only that of the mutated gene led to an increased basal level of NF-κB activation [37]. Similarly, overexpression of the p62^P392L mutant in HEK293 or Cos-1 cells increases basal and RANKL-induced NF-κB activation, more than overexpression of the wild-type p62 [71]. Finally, the presence of the PDB-associated p62^P392L mutation upregulates NFATc1 expression in pre-osteoclasts, favoring the increased osteoclastogenesis and osteoclast activity associated with metabolic bone disease [28,72]. These findings strongly suggest that p62^P392L contributes at least in part to the induction of an activated stage in osteoclasts by stimulating signaling pathways. More recent studies have further confirmed that CYLD knockdown significantly increased c-Fos expression in cells transduced to express both wild-type and mutant p62, without necessitating RANKL stimulation. Likewise, mutations to the UBA domain of p62 lead to a reduction in CYLD activity, and thus an increase in osteoclast development and resorption [72].

All of the mutations identified to date in PDB patients are clustered either within or near the C-terminal region of the p62 protein that embodies the ubiquitin-associated (UBA) domain, and affect its interactions with multiubiquitin chains, suggesting that an alteration of ubiquitin-chain binding by p62 is important in the development of PDB [73,74]. The accumulation of p62-associated proteins, due to impaired degradation through proteosomal or autophagic pathways, could explain some aberrant cellular functions. As p62 is a key player in selective autophagy, and may have a role in the formation of inclusion bodies [53,57], and because the inclusion bodies found in Pagetic osteoclasts resemble those observed in diseases with defective autophagy, a dysregulation of the autophagy process may well be part of the pathogeny in PDB, although so far no direct evidence has been provided for its role in the phenotype of Pagetic osteoclast [75]. In addition, the p62^P392L mutation did not affect the p62-related aggregate formation in human osteoclasts [4].

In osteoclast cultures derived from PDB patients it has been observed that lower rates of apoptosis are induced by the deprivation of survival factors or by death inducers, such as TRAIL, Fas activating antibody or TGF-β [37]. In CBMs cultures, apoptosis rates are lower in osteoclasts overexpressing either p62^wt or p62^P392L than in controls. Furthermore, it has been shown that p62 is overexpressed in PDB osteoclasts, and this could contribute to their resistance to apoptosis, given that increased expression of p62 could in itself have a protective effect against cell death by preventing the build-up of potentially cell-damaging proteins [56,76]. In addition, the basal and RANKL-induced activation of NF-κB may influence survival in PDB osteoclasts. Finally, some genes involved in osteoclast apoptosis (encoding Caspase-3, and TRAIL-R1) are downregulated in Pagetic osteoclasts [77], and the expression of anti-apoptotic Bcl-2 gene is upregulated [78]. So, while it is clear that apoptosis is hindered in PDB osteoclasts, and that the presence of PDB mutations in the p62 gene is involved in this, the mechanisms by which these apoptotic pathways are altered are still unknown.