Mutation of the sequestosome 1 (p62) gene increases osteoclastogenesis but does not induce Paget disease
Noriyoshi Kurihara, Yuko Hiruma, Hua Zhou, Mark A. Subler, David W. Dempster, Frederick R. Singer, Sakamuri V. Reddy, Helen E. Gruber, Jolene J. Windle, G. David Roodman
Noriyoshi Kurihara, Yuko Hiruma, Hua Zhou, Mark A. Subler, David W. Dempster, Frederick R. Singer, Sakamuri V. Reddy, Helen E. Gruber, Jolene J. Windle, G. David Roodman
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Research Article Bone biology

Mutation of the sequestosome 1 (p62) gene increases osteoclastogenesis but does not induce Paget disease

Abstract

Paget disease is the most exaggerated example of abnormal bone remodeling, with the primary cellular abnormality in the osteoclast. Mutations in the p62 (sequestosome 1) gene occur in one-third of patients with familial Paget disease and in a minority of patients with sporadic Paget disease, with the P392L amino acid substitution being the most commonly observed mutation. However, it is unknown how p62P392L mutation contributes to the development of this disease. To determine the effects of p62P392L expression on osteoclasts in vitro and in vivo, we introduced either the p62P392L or WT p62 gene into normal osteoclast precursors and targeted p62P392L expression to the osteoclast lineage in transgenic mice. p62P392L-transduced osteoclast precursors were hyperresponsive to receptor activator of NF-κB ligand (RANKL) and TNF-α and showed increased NF-κB signaling but did not demonstrate increased 1,25-(OH)2D3 responsivity, TAFII-17 expression, or nuclear number per osteoclast. Mice expressing p62P392L developed increased osteoclast numbers and progressive bone loss, but osteoblast numbers were not coordinately increased, as is seen in Paget disease. These results indicate that p62P392L expression on osteoclasts is not sufficient to induce the full pagetic phenotype but suggest that p62 mutations cause a predisposition to the development of Paget disease by increasing the sensitivity of osteoclast precursors to osteoclastogenic cytokines.

Authors

Noriyoshi Kurihara, Yuko Hiruma, Hua Zhou, Mark A. Subler, David W. Dempster, Frederick R. Singer, Sakamuri V. Reddy, Helen E. Gruber, Jolene J. Windle, G. David Roodman

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Figure 5

OCL formation and NF-κB gene reporter activity in OCL precursors from TRAP-p62P392L and WT mice.

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OCL formation and NF-κB gene reporter activity in OCL precursors from TR...
(A–C) OCL precursors (105 cells/well) from TRAP-p62P392L and WT mice were cultured in the presence of RANKL (A), TNF-α (B), or 1,25-(OH)2D3 (C). After 6 days of culture, cells were fixed and stained for TRAP activity. Results are expressed as mean ± SEM for quadruplicate cultures from a typical experiment. *P < 0.001 compared with results in WT cell cultures. Similar results were obtained in 5 independent experiments. Activation of NF-κB was measured by cotransfection of an NF-κB reporter plasmid and a β-gal expression vector into TRAP-p62P392L or WT OCL precursors following 24 hours of treatment with RANKL (D) or TNF-α (E). The results are expressed as the mean ± SEM for the ratio of NF-κB reporter activity to β-gal activity for quadruplicate determinations. *Significant differences (P < 0.001) compared with results with WT cultures. A similar pattern of results was seen in 2 independent experiments. (F) Six-month-old TRAP-p62P392L and WT mice were injected over the calvaria for 5 days with TNF-α (0, 0.375 μg, and 1.5 μg/day). Calvaria were harvested and tissue sections stained for TRAP activity (red color). Original magnification, ×100. (G) TRAP-positive OCL perimeter on the endosteal surface of calvaria in WT and TRAP-p62P392L mice. *Significant differences (P < 0.01) between TRAP-p62P392L and WT mice were found by 2-way ANOVA.