joint surfaces and in the primary spongiosa of the metaphyses were counted, and the number of c-Fos-positive osteoclasts was expressed as a percentage of the total number of osteoclasts at each site. IL-1 Protein Measurement—Culture medium from GFP- or c-Fos-infected cells grown on plastic or bone slices in 96-well plates or in the Transwell assay system was collected at the time when osteoclasts could be seen on the plastic of the culture wells or around bone slices using an inverted microscope (gen- erally after 7–9 days). A mouse IL-1 enzyme-linked immu- nosorbent assay kit (eBioscience, San Diego, CA) was used to measure IL-1 concentration according to the manufacturer’s instructions. Statistics—All results are given as the mean S.D. Compar- isons between two groups were analyzed using Student’s two- tailed unpaired t test. One-way analysis of variance and Dun- nett’s post hoc multiple comparisons were used for comparisons among three or more groups. p values 0.05 were considered statistically significant. Each experiment was repeated at least twice with similar results. RESULTS IL-1 Induces Osteoclast Formation Directly from Osteoclast Precursors Overexpressing c-Fos or NFATc1 and Synergizes with RANKL—To determine whether IL-1 can induce osteoclast formation from WT OCPs overexpressing c-Fos, we infected WT MDS with c-Fos retrovirus and cultured them on plastic culture plates in the presence of M-CSF. c-Fos-expressing MDS alone did not give rise to osteoclasts on plastic plates, but they formed numerous TRAP osteoclasts when they were treated with IL-1 (Fig. 1A). These cells also formed resorption pits when they were cultured on bone slices with IL-1 (data not shown). Furthermore, like TNF and RANKL (7), IL-1 induced osteoclast formation directly from NF- B dKO OCPs overexpressing c-Fos (Fig. 1A). In addition, subopti- mal doses of IL-1 and RANKL induced osteoclast formation synergistically (Fig. 1E). c-Fos induces expression of NFATc1, which is critical for osteoclastogenesis (12). We found that expression of NFATc1 was up-regulated in cells overexpressing c-Fos when they were treated with IL-1 for 72–96 h, the time when osteoclasts were forming in these cultures (data not shown). IL-1 induced oste- oclast formation from both WT and NF- B dKO MDS express- ing NFATc1 (Fig. 1B), and notably, NFATc1 induced osteoclast formation 1 day earlier than c-Fos, although both ultimately induced similar numbers of osteoclasts (data not shown). Overexpression of c-Fos in Osteoclast Precursors on Bone Slices Promotes Osteoclast Differentiation through Production of Soluble Factors—When we cultured c-Fos-expressing WT or NF- B dKO MDS on bone slices without IL-1, we found unex- pectedly that they gave rise to osteoclasts, and these cells also formed resorption pits (Fig. 1C, panels a–d, and D). Interest- ingly, osteoclasts also formed from c-Fos-expressing OCPs on the plastic around the bone slices (Fig. 1C, panels e and f). Because there was no other cytokine added to these bone slice cultures except M-CSF and because c-Fos-overexpressing OCPs cultured on plastic did not form osteoclasts in the absence of IL-1, we hypothesized that c-Fos-expressing OCPs grown on bone produce and release a soluble factor(s) to drive differentiation of the precursors on the adjacent plastic into mature osteoclasts or that osteoclasts or OCPs migrate from the bone onto the plastic. To examine whether c-Fos-expressing OCPs grown on bone secrete a soluble factor(s), we used a Transwell assay system. GFP- or c-Fos-expressing OCPs were cultured on bone slices in the upper chamber, and either GFP- or c-Fos-infected cells were cultured on plastic in the lower chamber of the Transwell culture system (Fig. 2A). These cultures also contained M-CSF. We found that c-Fos-expressing cells grown on the bone slices in the upper chamber induced mature osteoclasts from c-Fos- but not GFP-infected cells in the lower chamber (Fig. 2B). Fur- thermore, GFP-expressing OCPs cultured on bone slices in the upper chamber induced more osteoclasts in the lower cham- bers compared with c-Fos-expressing OCPs (Fig. 2B). Bone slices placed alone in the upper chamber without cells did not induce osteoclast formation from c-Fos-expressing OCPs. FIGURE 1. c-Fos overexpression in osteoclast precursors directly induces their differentiation into osteoclasts on bone slices. GFP, c-Fos, or NFATc1 retrovirus-infected WT and NF- B p50/p52 dKO MDS were cultured on plastic culture plates IL-1 or on bone slices M-CSF to form osteoclasts. Oste- oclasts were identified by TRAP staining, and resorption pits were visualized by toluidine blue staining. The number of osteoclasts (Ocls) that formed on plastic dishes from GFP- or c-Fos-infected (A) or NFATc1-infected (B) cells was assessed. TRAP osteoclasts in c-Fos-overexpressing cells on bone slices (C, panels a–d) and TRAP cells that formed on the plastic dish around bone slices (panels e and f) and the number of osteoclasts on bone slices and pit area (D) were assessed. c-Fos retrovirus-infected WT MDS were cultured with various doses of IL-1 and RANKL on plastic plates to form osteoclasts. The numbers of osteoclasts were assessed (E). Values are the mean S.D. of four plastic culture wells or bone slices. *, p 0.05 versus the respective value for GFP-infected cells or phosphate-buffered saline (PBS)-treated cells. IL-1 and c-Fos Induce Osteoclastogenesis through Bone Matrix APRIL 11, 2008•VOLUME 283•NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 9919 at SAECHSISCHE LA BIBL, on March 12, 2010 www.jbc.org Downloaded from

IL-1 Is Produced by Osteoclast Precursors Interacting with Bone and Promotes Osteoclastogenesis from c-Fos-expressing Precursors—TNF and IL-1 are pro-inflammatory cytokines known to be secreted by osteoclasts and OCPs (29). Thus, we considered both as candidate factors that could be produced by OCPs on bone slices to induce osteoclast formation. Both GFP- and c-Fos-expressing OCPs grown on bone slices expressed markedly more IL-1 mRNA (100–150-fold higher) compared with similar OCPs cultured on plastic dishes after 9 days of culture (Fig. 3A), the time when osteoclast formation begins. In contrast, the expression of TNF mRNA by the OCPs was increased by only 3–5-fold on bone slices. Furthermore, GFP- expressing OCPs expressed significantly higher levels of IL-1 mRNA on bone slices (Fig. 3A) and secreted more IL-1 protein into the culture medium compared with c-Fos-expressing OCPs (Fig. 3B). To determine whether IL-1 or TNF is responsible for the differentiation of c-Fos-expressing OCPs on bone slices and functions as a soluble factor, OCPs were infected with c-Fos retrovirus and cultured on bone slices in the presence of IL-1Ra or the TNF inhibitor ENBREL. IL-1Ra reduced c-Fos-induced osteoclast formation by 50% (Fig. 4A), an effect similar to that reported as the maximal effect of this protein to inhibit IL-1- induced osteoclast activity (29). In contrast, ENBREL at doses that completely blocked TNF-induced osteoclast formation on plastic had no inhibitory effect on c-Fos-induced osteoclast for- mation on bone slices (Fig. 4B). Furthermore, IL-1Ra reduced osteoclast formation (Fig. 4C) induced by IL-1 in c-Fos-ex- pressing OCPs cultured on plastic to a similar extent as in c-Fos-expressing OCPs without IL-1 on bone slices (Fig. 4A). Bone Matrix Proteins Stimulate Osteoclast Precursors to Pro- duce IL-1 and Induce Their Differentiation into Osteoclasts When c-Fos Is Overexpressed—Bone matrix contains at least 20 non-collagenous proteins, several of which have been reported to stimulate macrophages to produce cytokines such as IL-1 (43). To examine whether this is also the case for OCPs, we cultured OCPs on plastic, treated them with the bone matrix proteins (DSP, DPP, OPN, or TGF ), and determined their effects on the levels of IL-1 and TNF mRNA expression by real- time RT-PCR. DSP in particular and also OPN significantly increased IL-1 and to a lesser extent TNF mRNA expression, whereas DPP and TGF had no effect (Fig. 5A). To determine whether IL-1 induced by DSP or OPN can substitute for exogenous IL-1 and stimulate c-Fos-expressing OCPs to differentiate into osteoclasts, we treated GFP- or c-Fos-infected cells with DSP or OPN on plastic. As expected, these proteins did not induce osteoclast formation from GFP- infected cells. However, they induced osteoclast formation from c-Fos-expressing precursors, which was partially blocked by IL-1Ra (Fig. 5B) to the same extent as observed in cultures on bone slices (Fig. 4A) or in IL-1-induced osteoclast formation (Fig. 4C). TNF, but Not IL-1, Increases c-Fos Expression in Osteoclast Precursors—Our experiments demonstrated that IL-1-induced osteoclast formation requires forced c-Fos expression in OCPs. We reasoned that IL-1 would not have this effect by itself, oth- erwise IL-1 alone should be able to induce osteoclast formation. TNF has been reported to induce osteoclast differentiation FIGURE 2. Osteoclast precursors grown on bone slices induce osteoclast formation from c-Fos-overexpressing precursors on plastic below them. A, GFP or c-Fos retrovirus-infected WT MDS were cultured on bone slices or plastic in 24-well plates for 2 days in the presence of added M-CSF. Bone slices with either GFP- or c-Fos-infected cells or control slices with no cells were moved into the upper chambers of the Transwells and co-cultured with cells on plastic in the lower chambers as indicated in the presence of added M-CSF. B, the cells in the lower chambers were stained for TRAP activity, and the number of osteoclasts (Ocls) was counted. Values are the mean S.D. of three wells. *, p 0.05 versus the co-culture of c-Fos-infected cells in the lower chamber with blank bone. FIGURE 3. Osteoclast precursors grown on bone slices express markedly more IL-1 than those cultured on plastic plates. GFP or c-Fos retrovirus- infected WT MDS were cultured on plastic or bone slices in the presence of M-CSF for 9 days. A, total RNA was extracted and subjected to real-time RT- PCR to determine the expression levels of IL-1 and TNF. The -fold changes were calculated by dividing the value of the target gene by that of the GFP- infected samples on plastic. Values are the mean S.D. of triplicate loadings. *, p 0.05 versus cells grown on plastic. B, IL-1 protein concentrations were measured by enzyme-linked immunosorbent assay in the culture medium collected from the cultures described in A at the end of the culture. Data are the mean S.D. of five wells from one independent experiment representa- tive of three. *, p 0.05 between the two groups indicated. IL-1 and c-Fos Induce Osteoclastogenesis through Bone Matrix 9920 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 15• APRIL 11, 2008 at SAECHSISCHE LA BIBL, on March 12, 2010 www.jbc.org Downloaded from