A complementary DNA expression library derived from marrow samples from myeloma patients was recently screened and human macrophage inflammatory protein-1␣ (hMIP-1␣) was identified as an osteoclastogenic factor expressed in these samples. hMIP-1␣ enhanced osteoclast (OCL) formation in human marrow cultures and by highly purified OCL precursors in a dose-dependent manner (5-200 pg/mL). Furthermore, hMIP-1␣ enhanced OCL formation induced by human interleukin-6 (IL-6), which is produced by marrow stromal cells when they interact with myeloma cells. hMIP-1␣ also enhanced OCL formation induced by parathyroid hormone-related protein (PTHrP) and receptor activator of nuclear factor B ligand (RANKL), factors also implicated in myeloma bone disease. Timecourse studies revealed that the hMIP-1␣ acted during the last 2 weeks of the 3-week culture period. Reverse transcription-polymerase chain reaction analysis showed that the chemokine receptors for hMIP-1␣ (CCR1 and CCR5) were expressed by human bone marrow and highly purified early OCL precursors. Furthermore, hMIP-1␣ did not increase expression of RANKL. These data demonstrate that hMIP-1␣ is an osteoclastogenic factor that appears to act directly on human OCL progenitors and acts at the later stages of OCL differentiation. These data further suggest that in patients with myeloma, MIP-1␣ produced by myeloma cells, in combination with RANKL and IL-6 that are produced by marrow stromal cells in response to myeloma cells, enhances OCL formation through their combined effects on OCL precursors. (Blood. 2001;97:3349-3353)

Pagetic osteoclasts are greatly increased in number and size and have increased numbers of nuclei per cell compared to normal osteoclasts. The mechanisms responsible for enhanced osteoclast formation in Paget's disease are unknown. We have used our recently described model system for pagetic osteoclast formation to evaluate culture media conditioned by these atypical multinucleated cells (MNC) to determine if pagetic osteo-

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Summary Paget's Disease (PD) is characterized by abnormal osteoclasts (OCL) that secrete high IL-6 levels and induce exuberant bone formation. Because measles virus nucleocapsid gene (MVNP) and the p62P392L mutation are implicated in PD, marrows from 12 PD patients harboring p62P392L and 8 normals were tested for MVNP expression and pagetic OCL formation. 8/12 patients expressed MVNP and formed pagetic OCL in vitro, which were inhibited by antisense-MVNP. 4/12 patients lacked MVNP and formed normal OCL that were hyper-responsive to RANKL but unaffected by antisense-MVNP. Similarly, mice expressing only p62P394L formed normal OCL, while mice expressing MVNP in OCL, with or without p62P394L, developed pagetic OCL and expressed high IL-6 levels dependent on p38MAPK activation. IL-6 deficiency in MVNP mice abrogated pagetic OCL development in vitro. Mice co-expressing MVNP and p62P394L developed dramatic Paget's-like bone lesions. These results suggest that p62P394L and IL-6 induction by MVNP play key roles in PD.

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Activating transcription factor 4 (ATF4) is a critical transcription factor for osteoblast (OBL) function and bone formation; however, a direct role in osteoclasts (OCLs) has not been established. Here, we targeted expression of ATF4 to the OCL lineage using the Trap promoter or through deletion of Atf4 in mice. OCL differentiation was drastically decreased in Atf4 -/-bone marrow monocyte (BMM) cultures and bones. Coculture of Atf4 -/-BMMs with WT OBLs or a high concentration of RANKL failed to restore the OCL differentiation defect. Conversely, Trap-Atf4-tg mice displayed severe osteopenia with dramatically increased osteoclastogenesis and bone resorption. We further showed that ATF4 was an upstream activator of the critical transcription factor Nfatc1 and was critical for RANKL activation of multiple MAPK pathways in OCL progenitors. Furthermore, ATF4 was crucial for M-CSF induction of RANK expression on BMMs, and lack of ATF4 caused a shift in OCL precursors to macrophages. Finally, ATF4 was largely modulated by M-CSF signaling and the PI3K/AKT pathways in BMMs. These results demonstrate that ATF4 plays a direct role in regulating OCL differentiation and suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity.

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) 2 D 3 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.

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