A quest for clarity in bone erosion: The role of sequestosome 1 in Paget's disease of bone

Alterations in the SQSTM1 gene are a putative cause of Paget's disease of bone, yet results are conflicting about how these mutations impact osteoclasts, the cell type believed to be the main pathological contributor. In this issue of JBC, Zach et al. provide important new evidence that the protein encoded by SQSTM1, p62, negatively regulates osteoclastogenesis and demonstrate that aged p62–deficient mice develop bone phenotypes similar to those of Paget's disease. These findings help to clarify the role of this important protein and present new opportunities to interrogate bone biology.

mutations in p62 displayed increased size and nucleation (4 -6), along with activation of Nfatc1 and NF-B (7,8). Further discrepancies were seen in in vivo models of p62 deficiency. Mice generated with the equivalent of a human P392L mutation in SQSTM1 (the most frequent mutation in PDB) display a variety of phenotypes including decreased bone volume (5), local osteolytic lesions (6), or no phenotypic changes (4). So what does p62 really do?
Zach et al. (9) approached this question with similar strategies to past work, but with two important differences. First, they worked with a previously generated mouse model of p62/sequestosome 1 deficiency, which deleted exons 1-4 of the gene and had not been previously investigated for bone phenotypic changes. Second, they used a much reduced cell count in their in vitro differentiation protocols that avoids artificial inhibition of osteoclastogenesis. Consistent with previous findings, they found p62 was up-regulated during osteoclastogenesis in WT cultures. Furthermore, p62 deficiency led to an increase in osteoclast nucleation and differentiation and increased sensitivity to RANKL early in culture (i.e. days 3-4). However, no difference in osteoclast number was seen at later time points. These data led the authors to conclude that p62 is a negative regulator of osteoclastogenesis, differing from the findings of Duran et al. (3) but in agreement with other studies (4 -8). Interestingly, Zach et al. (9) found that p62 deficiency did not disrupt Nfatc1 or NF-B signaling, in contrast to two previous reports (7)(8). So, how does this regulation play out in vivo?
Zach et al. (9) next investigated the skeletal phenotype of p62-deficient mice, in which previous reports indicated varying degrees of bone volume change. Zach et al. (9) found that p62-deficient femurs were similar to WT femurs at 3 and 6 months, but there was an increase in bone mass at 9, 12, and 15 months. Histologically, p62-deficient mice had more mature osteoclasts in the lower femurs. Interestingly, closer examination of bones from 21-month-old mice revealed osteolytic lesions in the p62-deficient mice that are consistent with a PDB-like phenotype. Two serum markers of bone turnover were also significantly increased in p62deficient mice.
The work of Zach et al. (9) brings some clarity to the inconsistent findings about p62 in PDB, and importantly makes available an animal model that mimics human disease. Bone phenotypes depend highly on the mouse background strain, which may explain the differences between the mouse model used by Zach et al. (9) and those of previous reports. Additionally, due to a late onset of the PDB phenotype in the p62 mouse model of Zach et al. (9), models that previously did not show a phenotype, or showed local lesions (6), may develop lesions in older mice. The mice described in this work can now be used to delve deeper into the mechanisms underpinning PDB and may serve as a model for testing possible therapeutics.
The contributions of Zach et al. (9) also bring up important questions regarding the pathophysiology of PDB. First, what is the role of p62 in osteoclast differentiation? Monocytes and macrophages are given cues that initiate their transformation into osteoclasts. Zach et al. (9) showed an up-regulation of p62 in culture conditions that promote macrophages relative to in osteoclast cultures, so p62 may maintain monocytes in their more naïve form. This suggests that in the absence of p62, monocyte differentiation is less regulated and an increase in osteoclast differentiation is seen (Fig. 1). It will be interesting to see if this holds true in vivo. Second, what about other cells in the bone marrow microenvironment? Although much of the focus of PDB research is on osteoclasts, osteoblasts are also affected by p62 mutations. Osteoblasts deficient in p62 produce more RANKL, which stimulates osteoclast differentiation (4). Other cells also regulate osteoblast and osteoclast differentiation and activity via secreted factors and direct cell-to-cell contact. For example, Chang et al. (10) found that osteoblast-specific depletion of p62 resulted in low bone mass due to impaired macrophage-dependent osteoblast differentiation. Understanding the role of p62 in cells of the marrow compartment may help uncover non-cell autonomous effects on osteoclasts and osteoblasts. We look forward to these answers and more, as the quest for insights into PDB continues. Coupling of bone formation and resorption maintains normal bone levels. When p62 is mutated or deleted, the negative regulation of osteoclast differentiation is lost and osteoclast number, size, and nucleation increase, resulting in osteolytic lesions (right panel). Due to coupling of resorption and formation, osteoblasts respond to form new, highly disorganized bone in the lesions. In the absence of p62, osteoblasts also increase RANKL secretion, further enhancing osteoclast formation.