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 p62^P392L mutation contributes to the development of this disease. To determine the effects of p62^P392L expression on osteoclasts in vitro and in vivo, we introduced either the p62^P392L or WT p62 gene into normal osteoclast precursors and targeted p62^P392L expression to the osteoclast lineage in transgenic mice. p62^P392L-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, TAF_II-17 expression, or nuclear number per osteoclast. Mice expressing p62^P392L 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 p62^P392L 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.

Paget disease (PD) is the second most common bone disease in persons of Anglo-Saxon descent over the age of 55 (1). It is the most exaggerated example of disordered bone remodeling, with abnormalities in all phases of the bone remodeling process (2). The primary cellular abnormality resides in the osteoclast (OCL). OCLs in PD are increased in number and size and have increased numbers of nuclei (3). In addition, they are hyperresponsive to 1,25-(OH)₂D₃ and receptor activator of NF-κB ligand (RANKL) (4, 5) and show increased expression of TAF_II-17, a member of the TF_IID transcription complex, which acts as a coactivator of vitamin D receptor–mediated transcription and is upregulated in pagetic osteoclasts (6).

Both genetic and environmental factors have been proposed as contributing to the etiology of PD, and multiple families with an autosomal dominant mode of inheritance have been described (7, 8). Recently, mutations in the p62 (sequestosome 1) gene have been linked to approximately 30% of patients with familial PD and to a minority of patients with sporadic PD (8). All of the mutations identified to date lie within or near the ubiquitin-binding domain in the carboxyterminal region of the protein, with the P392L amino acid substitution representing the most frequently observed mutation (8). p62 plays a critical role in NF-κB activation induced by TNF-α, CD40, and IL-1 through its interactions with the atypical protein kinases ζPKC and λPKC (9, 10). However, the role of p62 mutations in PD is unclear since not all individuals carrying a p62 mutation have PD (11–13).

It is our hypothesis that both genetic and nongenetic factors are required for the development of PD and that the genetic factors, such as p62 mutations, function to increase OCL formation but are not sufficient to induce the abnormal OCLs or pagetic bone lesions characteristic of PD. To test this hypothesis, we characterized OCL precursors from Paget patients carrying the p62^P392L mutation as well as control human OCL precursors transduced with either p62 or p62^P392L expression vectors. We also determined whether the p62^P392L gene can induce pagetic-like OCLs or bone lesions in vivo when expressed in OCL precursors of transgenic mice.

OCL precursors from PD patients carrying the p62^P392L gene are hyperresponsive to RANKL, TNF-α, and 1,25-(OH)₂D₃ and express increased levels of TAF_II-17. OCL precursors from PD patients carrying the p62^P392L mutation or from controls were compared for their capacity to form OCLs over a range of concentrations of RANKL, TNF-α, and 1,25-(OH)₂D₃. OCL precursors from patients carrying the p62^P392L mutation were hyperresponsive to RANKL and TNF-α as well as 1,25-(OH)₂D₃ (Figure 1, A–C); this is similar to previous findings from patients with sporadic PD (3–5). In cultures of OCL precursors from PD patients with p62^P392L mutations, maximum OCL formation was obtained at RANKL and TNF-α concentrations of 1–10 ng/ml and 1–10 pg/ml, respectively, compared with 100 ng/ml and 50 pg/ml for normal OCL precursors. Similar results were obtained with 3 individual PD patients. Further, the OCLs that formed contained increased numbers of nuclei per OCL (Table 1) and elevated expression of TAF_II-17 (Figure 1D). However, measles virus nucleocapsid protein (MVNP) transcripts were also present in OCLs from these patients (Figure 1D), raising the possibility that a subset of the phenotypic characteristics of these OCLs might be caused by the presence of MVNP rather than p62^P392L.

Figure 1

OCL formation and expression of MVNP and TAF_II-17 in GM-CFU from p62^P392L-positive PD patients and controls. (A–C) GM-CFU (10⁵ cells/well) from Paget patients known to harbor the p62^P392L mutation and from normal individuals were cultured for OCL formation in the presence of RANKL (A), TNF-α (B), or 1,25-(OH)₂D₃ (C). After 3 weeks of culture, cells were fixed and stained with the 23c6 monoclonal antibody, which identifies OCLs. Results are expressed as the mean ± SEM for quadruplicate determinations. *Significant differences (P < 0.001) compared with results from cultures of normal GM-CFU treated with the same concentration of each factor. A similar pattern of results was seen in 2 independent experiments. Paget 1 and Paget 2 refer to PD patient sample groups 1 and 2. MNC, multinuclear cell. (D) RT-PCR analysis of MVNP and TAF_II-17 mRNA expression in GM-CFU from p62^P392L-positive PD patients and controls cultured for 2 days with 1,25-(OH)₂D₃. RT-PCR analysis of β-actin expression was included as a control for mRNA quality and amplification.

Table 1

Nuclear number per OCL

OCL formation by p62- and p62^P392L-transduced normal OCL precursors. To further dissect the role of p62^P392L in PD, we transduced normal human OCL precursors with the WT p62 or p62^P392L gene or empty vector (EV). The total p62 expression levels were determined by immunoblotting extracts from transduced GM-CFU–derived cells with an antibody that recognizes both WT and mutant p62. The p62 protein was detected in both p62- and p62^P392L-transduced GM-CFU without treatment with RANKL. In contrast, p62 protein was only detected in EV-transduced GM-CFU after 1 day of treatment with RANKL (data not shown).

p62^P392L-transduced OCL precursors treated with varying concentrations of RANKL or TNF-α were found to be hyperresponsive to both cytokines and formed increased numbers of OCLs (Figure 2, A and B). While overexpression of WT p62 resulted in a degree of hyperresponsivity to RANKL similar to that of p62^P392L, the p62^P392L cells were much more hyperresponsive to TNF-α than WT p62 cells. Both p62- and p62^P392L-transduced OCL precursors formed significantly larger OCLs than EV cells (Figure 2D), although nuclear number per OCL was not increased in p62- or p62^P392L-derived OCLs regardless of treatment (Table 1). Also, a 7-fold increase in bone resorption was observed when OCLs formed by p62^P392L-transduced human GM-CFU were treated with RANKL (50 ng/ml) and cultured on dentin, as compared with RANKL-treated EV-transduced OCLs (Figure 3, A and B).

Figure 3

Resorption lacunae formed by OCLs from p62^P392L- and EV-transduced human GM-CFU. (A) Resorption lacunae formed on dentin by OCL. Original magnification, ×100. (B) Resorption areas per dentin slice for each treatment group. Results represent mean ± SEM for quadruplicate determinations for a typical experiment. Similar results were seen in 3 independent experiments. *Significant differences (P < 0.05).

In contrast with the results with RANKL and TNF-α, neither p62- nor p62^P392L-transduced OCL precursors were hyperresponsive to 1,25-(OH)₂D₃ (Figure 2C) or formed increased numbers of OCLs compared with EV-transduced cells. In addition, neither p62- nor p62^P392L-transduced GM-CFU–derived cells showed elevated expression of TAF_II-17 in the presence or absence of 10^–10 M 1,25-(OH)₂D₃ (Figure 4).

Figure 4

TAF_II-17 expression in p62-, p62^P392L-, and EV-transduced human OCL precursors. GM-CFU–derived cells (10⁶ cells) transduced with p62, p62^P392L, and EV were cultured for 2 days with 10^–8 M 1,25-(OH)₂D₃. RNA was then prepared and subjected to RT-PCR analysis of TAF_II-17 or β-actin expression. The 2 PD patient samples shown in Figure 1 were used as positive controls.

Effects of p62^P392L expression on OCLs in vivo. Since OCLs harbor the primary cellular abnormality in PD, we used the tartrate-resistant acid phosphatase (TRAP) promoter to target expression of the human p62^P392L gene to the OCL lineage in transgenic mice (TRAP-p62^P392L mice) to determine the effects of p62^P392L expression in OCLs in vivo. Eight lines of TRAP-p62^P392L transgenic mice were generated from independent founder mice, and each of these was characterized with regard to transgene expression level, TNF-α responsivity, and histology. The results described below were obtained from a single line (Tp62m2), although similar results were observed in multiple other lines as well. The expression levels of total p62 protein in mice of the Tp62m2 line, as determined by immunoblotting of extracts from bone marrow cells with an antibody that detects both murine and human p62, were found to be approximately 2.5-fold higher than p62 levels in WT mice (data not shown).

Histomorphometric evaluation of vertebral bones from TRAP-p62^P392L mice at 4, 8, 12, and 16 months of age revealed an increase in OCL perimeter (the amount of bone surface covered with TRAP-positive, mono-, and multinuclear cells) and a progressive reduction in cancellous bone volume when compared with that of age-matched WT controls (Table 2 and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI28267DS1). The decrease in cancellous bone volume was associated with decreases in both trabecular width and number. Although OCL perimeter was elevated, there was no coupled increase in osteoblast perimeter, as is seen in PD lesions.

Table 2

Cancellous bone structure and bone turnover in TRAP-p62^P392L mice

Electron microscopic examination of OCLs from TRAP-p62^P392L mice demonstrated that the cells did not contain the nuclear inclusions characteristic of pagetic OCLs. OCLs were similar to those in WT controls in terms of nuclear and cytoplasmic ultrastructure as well in the morphology of the ruffled border (data not shown).

OCL precursors from TRAP-p62^P392L mice are hyperresponsive to RANKL and TNF-α but not 1,25-(OH)₂D₃. When marrow cells from TRAP-p62^P392L mice were cultured with RANKL and TNF-α to induce OCL formation, they were found to be hyperresponsive to both cytokines, and they formed increased numbers of OCLs as compared with nontransgenic littermates (Figure 5, A and B). Similarly, treatment of TRAP-p62^P392L mice with TNF-α (0–1.5 μg/d) significantly increased OCL formation compared with that of WT mice at all concentrations tested (Figure 5, E and G). Further, the dose-response curves for NF-κB reporter gene activity in TRAP-p62^P392L OCL precursors for both RANKL and TNF-α were shifted to the left compared with cells from WT mice (Figure 5, D and E). However, OCL precursors from TRAP-p62^P392L mice were not hyperresponsive to 1,25-(OH)₂D₃ (Figure 5C) and did not express detectable TAF_II-17 (data not shown). They also did not have increased nuclear number per OCL (Table 1). These results are consistent with our results from p62^P392L-transduced human OCL precursors and indicate that expression of p62^P392L induces a subset of pagetic characteristics, including hyperresponsivity to the osteoclastogenic cytokines RANKL and TNF-α, but does not result in development of OCLs that express the complete pagetic phenotype or development of pagetic-like lesions.

Figure 5

OCL formation and NF-κB gene reporter activity in OCL precursors from TRAP-p62^P392L and WT mice. (A–C) OCL precursors (10⁵ cells/well) from TRAP-p62^P392L 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-p62^P392L 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-p62^P392L 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-p62^P392L mice. *Significant differences (P < 0.01) between TRAP-p62^P392L and WT mice were found by 2-way ANOVA.

To further examine the mechanisms responsible for the increased levels of OCL formation in marrow cultures from TRAP-p62^P392L mice, we determined the time course for OCL formation, the rates of proliferation of OCL precursors and of OCL apoptosis, and the expression levels of OCL differentiation markers. As shown in Figure 6A, OCL formation in marrow cultures from TRAP-p62^P392L mice was maximum after 6 days of culture while OCL formation in WT cultures was maximum at 9 days of culture. Further, OCL precursor proliferation was significantly increased in marrow cultures from p62^P392L mice compared with WT cultures but followed a similar time course (Figure 6B). In contrast, although the number of apoptotic OCLs was increased in TRAP-p62^P392L marrow cultures compared with WT marrow cultures, the percentages of apoptotic OCLs were similar (18% versus 10%; P > 0.05) in TRAP-p62^P392L and WT cultures after 9 days (Figure 6C). Apoptotic OCLs were not detected in cultures of mice from either genotype at day 3 or 6 of culture. Marrow cultures from TRAP-p62^P392L mice expressed relatively higher levels of TRAP, cathepsin K, and calcitonin receptor mRNA compared with WT cultures (Figure 7).

Figure 6

Time course for OCL formation, OCL precursor proliferation, and OCL apoptosis by marrow cells from TRAP-p62^P392L or WT mice. (A) OCL precursors (10⁵ cells/well) from TRAP-p62^P392L and WT mice were cultured in the presence of RANKL or TNF-α. After 3, 6, or 9 days of culture, cells were fixed and number of TRAP-positive OCLs counted. (B) OCL precursors (1 × 10⁶ per culture) from WT (white bars) and TRAP-p62^P392L (black bars) littermate mice were cultured for 2, 4, or 6 days and were pulsed at the end of the culture period for 1 hour with 1 mCi [³H]-thymidine. Radioactivity was counted by liquid scintillation spectrometry. Results are expressed as the mean ± SD for quadruplicate cultures. *P < 0.001 compared with results of WT cultures treated with the same concentration of RANKL or TNF-α. (C) OCL precursors (10⁵ cells/well) from TRAP-p62^P392L and WT mice were cultured in the presence of RANKL or TNF-α. After 9 days of culture, cells were fixed and number of apoptotic OCLs were counted using a commercial Annexin V kit (Promega). All results are expressed as the mean ± SD for quadruplicate cultures.

Figure 7

Expression of markers of OCL differentiation by TRAP-p62^P392L and WT OCL precursors. OCL precursors (10⁶ cells) from WT and TRAP-p62^P392L littermates were cultured for 2, 4, or 6 days with 50 ng/ml of RANKL or 100 pg/ml TNF-α. RNA was prepared and subjected to RT-PCR analysis for cathepsin K, MMP-9, calcitonin receptor, and TRAP as described in Methods. Ratio of marker mRNA expression to β-actin is shown below each lane. Lane 1, WT M-CSF (10 ng/ml); lane 2, WT M-CSF (10 ng/ml) plus RANKL (50 ng/ml); lane 3, WT M-CSF (10 ng/ml) plus TNF-α (100 pg/ml); lane 4, p62^P392L M-CSF (10 ng/ml); lane 5, p62^P392L M-CSF (10 ng/ml) plus RANKL (50 ng/ml); lane 6, p62^P392L M-CSF (10 ng/ml) plus TNF-α (100 pg/ml).

Since OCL precursors from Paget patients carrying the P392L mutation were hyperresponsive to 1,25-(OH)₂D₃ and also expressed MVNP, we transfected OCL precursors from TRAP-p62^P392L mice with MVNP and determined their responsivity to 1,25-(OH)₂D₃. As shown in Figure 8, OCL precursors from TRAP-p62^P392L mice transfected with MVNP were hyperresponsive to 1,25-(OH)₂D₃ and formed OCL at concentrations that were 1 to 2 logs lower than those of EV-transfected cells.

Figure 8

MVNP-transduced TRAP-p62^P392L mouse GM-CFU cells are hyperresponsive to 1,25-(OH)₂D₃. OCL precursors (5 × 10⁵ cells/well) from MVNP- or EV-transduced TRAP-p62^P392L mouse GM-CFU were cultured in the presence of 1,25-(OH)₂D₃. After 9 days of culture, cells were fixed and stained for TRAP activity. Results are expressed as the mean ± SEM for quadruplicate cultures. *P < 0.001 compared with results of cultures of EV-transduced GM-CFU treated with the same concentration of 1,25-(OH)₂D3.

To further delineate the mechanisms responsible for the enhanced OCL formation in TRAP-p62^P392L mice, we examined inhibitor of NF-κB (IκB), p38 MAPK, and extracellular signal–regulated kinase 1/2 (ERK1/2) signaling in nonadherent marrow cells from WT and TRAP-p62^P392L mice. As shown in Supplemental Figure 2A, p-ERK1/2 was increased in marrow cells treated with RANKL or TNF-α while only modest changes were seen in p38 MAPK and p-IκB activity. Transfection of GM-CFU from WT and TRAP-p62^P392L mice with the MVNP gene further increased levels of NF-κB in TRAP-p62^P392L OCL precursors compared with those of WT precursors (Supplemental Figure 2B). Expression of MVNP in OCL precursors from WT or TRAP-p62^P392L mice did not increase expression of c-Fos.

Mutations in the p62 gene have been linked to PD in approximately one-third of patients with familial PD and a minority of patients with sporadic PD. However, it is unlikely that these mutations are sufficient to induce the OCL abnormalities and bone lesions that are characteristic of PD, since pagetic lesions are focal even in patients carrying germline p62 mutations and some individuals harboring p62 mutations fail to develop PD (11–13). To determine the role of p62 mutation in PD, we characterized OCL precursors from familial PD patients carrying the most common PD-associated p62 mutation (P392L) and compared them with normal OCL precursors. OCL precursors from p62^P392L-positive PD patients formed OCLs at lower concentrations of RANKL, TNF-α, and 1,25-(OH)₂D₃ than controls (Figure 1, A–C) and had increased TAF_II-17 expression (Figure 1D), all characteristic of PD; this was similar to our previous findings in OCL precursors from patients with sporadic PD (3–6). However, OCL precursors from these patients also expressed MVNP, which we have previously reported results in increased OCL formation in response to RANKL and 1,25-(OH)₂D₃ and to increased expression of TAF_II-17 both in vitro and in vivo (6, 14).

To further characterize the contributions of p62^P392L to PD in the absence of MVNP, we transduced normal human OCL precursors with vectors encoding WT p62 or p62^P392L or with EV. As with OCL precursors from PD patients, p62^P392L-transduced OCL precursors showed increased TNF-α and RANKL sensitivity (Figure 2, A and B) but, in contrast, did not demonstrate increased 1,25-(OH)₂D₃ responsivity (Figure 2C) or increased expression of TAF_II-17 (Figure 4). In addition, nuclear number per OCL was not increased in p62^P392L-transduced OCLs (Table 1). Thus, these OCLs express only a subset of the characteristics of pagetic OCLs (15). Similar results were obtained when expression of the p62^P392L gene was targeted to cells in the OCL lineage in vivo. OCL precursors from TRAP-p62^P392L mice were hyperresponsive to RANKL and TNF-α but not to 1,25-(OH)₂D₃ (Figure 5, A–C) and did not express increased levels of TAF_II-17. Further, OCL formation in TRAP-p62^P392L mice was significantly increased in vivo by treatment with TNF-α (Figure 5, F and G).

Both RANKL and TNF-α activate signaling pathways involving p62 that ultimately lead to the activation of NF-κB, p38 MAPK, and ERK1/2, which are important for OCL formation and function. Our results demonstrate elevated NF-κB activation as well as p38 MAPK and ERK1/2 signaling in OCLs expressing p62^P392L, strongly suggesting that Paget-associated mutations in p62 lead to increased osteoclastogenesis by stimulating signaling pathways that activate NF-κB (Supplemental Figure 2A). However, the detailed mechanisms by which these p62 mutations activate signaling remain to be determined. All of the PD-associated p62 mutations reside in or near the ubiquitin-binding domain and result in loss of the ubiquitin-binding capacity of p62 (16). Thus, it is possible that the ubiquitin-binding domain of p62 normally mediates a protein/protein interaction that dampens NF-κB signaling in OCLs in response to inflammatory cytokines, such that loss of this interaction leads to increased activation of these pathways.

It is interesting to note that in both transduced human OCL precursors and in transgenic mouse OCL precursors, expression of p62^P392L had a much more dramatic effect on responsivity to TNF-α than to RANKL. Consistent with our results, Duran et al. have reported that the P392L mutation in p62 increased NF-κB reporter activity (17). These results suggest that TNF-α, in addition to RANKL and 1,25-(OH)₂D₃, may be involved in the increased osteoclastogenesis in PD and should be studied further.

Histomorphometric analysis of vertebral cancellous bone in the TRAP-p62^P392L transgenic mice revealed a phenotype that was characterized by low bone volume with reduced trabecular number and width. OCL perimeter was increased at all ages examined, but there was no coupled increase in osteoblast perimeter. This marked imbalance between OCL formation and new bone formation is analogous to imbalance in inflammatory diseases of bone, such as rheumatoid arthritis and lytic bone metastases, in which bone formation is suppressed, rather than PD, in which OCL activity is closely coupled to new bone formation. This phenotype is very different from that observed in TRAP-MVNP transgenic mice, which persistently express the gene encoding the MVNP (14). In contrast with TRAP-p62^P392L mice, TRAP-MVNP mice displayed a bone phenotype that closely resembled PD in humans. This included a coupled increase in both bone resorption and new bone formation and enlarged OCLs with increased nuclear number (14). Moreover, a subset (30%) of 12-month-old TRAP-MVNP mice displayed pagetic-like lesions, with increased bone volume and dramatically thickened and disorganized trabeculae composed primarily of woven bone. No such lesions were observed in the TRAP-p62^P392L mice, which were examined up until 18 months of age. In addition, OCL precursors from the TRAP-MVNP mice showed increased sensitivity to 1,25-(OH)₂D₃ and expressed increased levels of TAF_II-17, findings that were not observed in the TRAP-p62^P392L mice unless the cells were transfected with MVNP (Figure 8). Finally, transfection of OCL precursors from TRAP-p62^P392L mice with MVNP further increased levels of NF-κB, suggesting that MVNP can increase the enhanced OCL formation induced by the p62^P392L mutation (Supplemental Figure 2B).

One possible explanation for the failure of the TRAP-p62^P392L mice to display increased osteoblast activity is that expression of p62^P392L is restricted to cells of the OCL lineage in this model while familial PD patients carrying a p62 mutation express the mutant protein in all cell types. It remains to be determined whether the p62 mutation plays a direct role in other cell types besides OCL in PD. It will therefore be of interest to evaluate the bone phenotype in knockin mice carrying the analogous p62 mutation in the germline, which are currently being developed.

Taken together, these results demonstrate that the expression of p62^P392L in OCLs increases OCL formation but is not sufficient to induce PD. Thus, p62 mutations may predispose to the development of PD by increasing basal OCL activity, but 1 or more additional factors are required for development of the full PD phenotype.

View Supplemental data

This work was supported by research funds from the NIH grant PO1-AR049363 and US Army Medical Research & Materiel Command (USAMRMC) award DAMD17-03-1-0763 and is the result of work supported with resources and the use of facilities at the VA Pittsburgh Healthcare System, Research and Development. We acknowledge the General Clinical Research Center at the University of Pittsburgh for assistance with obtaining the marrow samples and the VCU Massey Cancer Center L.T. Christian III Transgenic Mouse Core Facility, which is supported by NIH grant P30-CA16059. J.J. Windle wishes to thank Heju Zhang and Christina Boykin for assistance with transgenic mouse production. H.E. Gruber wishes to thank Daisy Ridings for expert assistance with electron microscopy.

Address correspondence to: Noriyoshi Kurihara, VA Pittsburgh Healthcare System, Research and Development (151C-U), University Drive C, Pittsburgh, Pennsylvania 15240, USA. Phone: (412) 688-6000 ext. 81-4990; Fax: (412) 688-6960; E-mail: kuriharan@upmc.edu.

Nonstandard abbreviations used: ERK1/2, extracellular signal–regulated kinase 1/2; EV, empty vector; IκB, inhibitor of NF-κB; MVNP, measles virus nucleocapsid protein; OCL, osteoclast; PD, Paget disease; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate-resistant acid phosphatase.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.117:133–142 (2007). doi:10.1172/JCI28267

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