A SQSTM1/p62 mutation linked to Paget’s disease increases the osteoclastogenic potential of the bone microenvironment

Abstract

Paget's disease of bone (PDB) is the second most common bone disease and is characterized by focal bone lesions which contain large numbers of abnormal osteoclasts (OCLs) and very active normal osteoblasts in a highly osteoclastogenic marrow microenvironment. The etiology of PDB is not well understood and both environmental and genetic causes have been implicated in its pathogenesis. Mutations in the SQSTM1/p62 gene have been identified in up to 30% of Paget's patients. To determine if p62 mutation is sufficient to induce PDB, we generated mice harboring a mutation causing a P-to-L (proline-to-leucine) substitution at residue 394 (the murine equivalent of human p62(P392L), the most common PDB-associated mutation). Bone marrow cultures from p62(P394L) mice formed increased numbers of OCLs in response to receptor activator of NF-kappaB ligand (RANKL), tumor necrosis factor alpha (TNF-alpha) or 1alpha,25-(OH)(2)D(3), similar to PDB patients. However, purified p62(P394L) OCL precursors depleted of stromal cells were no longer hyper-responsive to 1alpha,25-(OH)(2)D(3), suggesting effects of the p62(P394L) mutation on the marrow microenvironment in addition to direct effects on OCLs. Co-cultures of purified p62(P394L) stromal cells with either wild-type (WT) or p62(P394L) OCL precursors formed more OCLs than co-cultures containing WT stromal cells due to increased RANKL production by the mutant stromal cells. However, despite the enhanced osteoclastogenic potential of both OCL precursors and marrow stromal cells, the p62(P394L) mice had histologically normal bones. These results indicate that this PDB-associated p62 mutation is not sufficient to induce PDB and suggest that additional factors acting together with p62 mutation are necessary for the development of PDB in vivo.

Measles virus nucleocapsid protein increases osteoblast differentiation in Paget’s disease

“…In contrast, mice with germline transmission of the mouse equivalent of the most frequent mutation linked to PD, p62 P394L , did not develop pagetic-like bone lesions (12,13). These results suggested that MVNP expression in OCLs was driving the increased local bone formation and that results from these mice should inform the factors driving increased bone formation in patients with PD.…”

“…To determine whether ephrinB2 was increased in PD patients, we tested OCL precursors from PD patients who harbored the p62 P392L mutation and did or did not express MVNP (12,13,23). To circumvent the difficulty of obtaining marrow samples from PD patients, we assayed ephrinB2 expression in OCL precursors isolated from the peripheral blood of PD patients who we previously confirmed did or did not express MVNP in their marrow-derived OCLs and compared these OCL precursors with those from 2 age-matched controls.…”

“…Transgenic mice expressing MVNP under the control of the mouse TRACP promoter were previously described (20), as were KI mice carrying a proline-to-leucine substitution at residue 394 of the endogenous mouse SQSTM1/p62 gene (equivalent to human p62 P392L ) (12). All data are from mice generated from this p62 P394L MVNP colony (not from the parental p62 P394L or MVNP colonies).…”

“…However, mutations like p62 P392L are apparently insufficient to induce PD, since some individuals harboring p62 mutations never develop the disease (10). Further, mice with a germline p62 P394L mutation (p62-knockin [p62-KI] mice), the murine equivalent of human p62 P392L , exhibit increased OCL numbers but do not develop the markedly enhanced bone formation characteristic of PD (12). These results suggest that genetic factors increase OCLs, but environmental or other factors are also required for the increased bone formation that occurs in PD.…”

Role of NF-κB in the skeleton

“…How p62 and the autophagy process might interact in the OC in the context of NF-κB signaling is yet to be determined. Knock-in mice bearing a PD p62 mutation (P394L) have OCs with increased responsivity to RANKL and low bone mass, but lack the focal lesions and hypersensitivity to vitamin D3 seen in human PD [87], suggesting that other factors are required for PD. The measles protein, MVNP, when expressed in OC precursors, recapitulates the PD features such as focal lesions and hypersensitivity to vitamin D3, which is not produced by p62 mutation [88], but there is no evidence that these effects are mediated by NF-κB.…”