Osteoclasts are multinucleated giant cells derived from the monocyte/macrophage lineage. Their differentiation is triggered by RANKL in the presence of macrophage colony-stimulating factor (M-CSF, encoded by Csf-1
gene), which is produced by osteoblasts1,2
. RANKL induces intracellular signals via its receptor RANK, and upregulates the expression of various genes, such as Nfatc1,fos, Oscar, Ctsk
, and Calcr
that encode proteins for NFATc1, c-Fos, OSCAR, cathepsin K and calcitonin receptor, respectively3–5
. Numerous studies have focused on these upregulated genes and their roles in osteoclastogenesis. On the other hand, the expression levels of various genes are simultaneously downregulated during osteoclastogenesis6
. The biological significance of the downregulated expression of these genes following RANK activation, however, has not been fully elucidated.
To identify genes that show reduced expression levels in response to RANK signaling, we performed a genome-wide screening of mRNAs from osteoclast precursors and osteoclasts using a DNA microarray technique (data not shown). Among the identified genes, expression of the transcription factor Irf8
[also called interferon consensus sequence binding protein (ICSBP)] was found to be downregulated during the initial phase of osteoclastogenesis triggered by RANKL (data not shown). IRF8 is known to be specifically expressed in immune cells, including monocytes/macrophages, B lymphocytes, and activated T lymphocytes7–9
. It is a member of the IRF family and has been shown to regulate myeloid cell development by interacting with the Ets family transcription factors10,11
. Hence, Irf8–/–
mice show an increased number of myeloid progenitors, defects in macrophage function including impaired IL-12 production, developmental defects in CD8α+
and plasmacytoid dendritic cells, and systemic expansion of the granulocyte population, which frequently leads to a fatal blast crisis12–14
Using RT-PCR and immunoblot analysis, IRF8 expression was detected in bone marrow-derived macrophages (BMMs) and spleen-derived macrophages, which are capable of differentiating into osteoclasts (). As previously shown3
, NFATc1 was modestly expressed at a basal level in unstimulated BMMs and NFATc1 expression was strongly induced after RANKL stimulation (); robust induction of NFATc1 by RANKL is a necessary and pivotal step for osteoclast differentiation characterized by enhanced expression of osteoclast marker genes such as Oscar, Itgb3
that encodes integrin β3 protein, and Ctsk3
. In contrast, IRF8 expression decreased after RANKL stimulation (), resulting in substantially lower nuclear IRF8 protein 24 h after RANKL addition (). We hypothesized that IRF8 downregulation may be required for osteoclast differentiation and that IRF8 may inhibit early stages of osteoclastogenesis.
Figure 1 IRF8 inhibits osteoclastogenesis in BMMs stimulated with M-CSF and RANKL. (a) mRNA expressions of Irf8, Nfatc1, Oscar, Itgb3, Ctsk and Gapdh in BMMs 0, 6, 12, 24, 48 and 72 h after M-CSF (50 ng/ml) and RANKL (150 ng/ml) stimulation (RT-PCR). (b) Immunoblot (more ...)
To examine the role of IRF8 in osteoclastogenesis, we constructed a retroviral vector, pMX-Irf8-IRES-EGFP, which was engineered to express both IRF8 and enhanced green fluorescence protein (EGFP). Macrophage-like osteoclast precursors were transduced with retroviral particles encoding Irf8 or control viral particles that lacked the Irf8 sequence (pMX-IRES-EGFP). IRF8–overexpressing precursors failed to differentiate into osteoclasts in response to RANKL stimulation (). Furthermore, these cells were able to phagocytize zymosan particles, whereas multinucleated osteoclasts failed to do so (). These results suggest that IRF8 inhibits osteoclastogenesis from precursor cells, which instead retain the characteristics of phagocytic macrophages.
Next, we analyzed the Irf8–/–
mice for abnormal bone phenotypes. Radiographic and microcomputed tomographic analyses showed that these mice had severe osteoporosis accompanied by dramatic decreases in trabecular bone volume, number, and thickness, as well as the number of bone nodules (). Histomorphometric analysis also revealed reduced bone mass in the Irf8–/–
mice (data not shown). Importantly, increases in the osteoclast number and surface area were observed in these mice (). There were no significant differences between wild-type and Irf8–/–
mice in the serum level of osteoprotegerin (OPG encoded by Tnfrsf11b
gene), a decoy RANKL receptor that inhibits osteoclastogenesis, or in RANKL levels in primary cultured osteoblasts (Supplementary Fig. 1
online). Moreover, an increased rate of bone formation accompanying the accelerated bone resorption rate was observed in the Irf8–/–
mice, suggesting that their osteoporosis was caused by enhanced bone turnover and remodeling (). Together, these observations indicated that IRF8 plays a suppressive role in osteoclastogenesis during in vivo
bone remodeling. To address whether the bone phenotype could be explained by Irf8
deficiency in osteoclast precursors, or whether a cell autonomous effect of Irf8
deficiency in osteoblasts or bone marrow stromal cells could play a role, we established chimeric mouse models in which either wild-type or Irf8–/–
littermate bone marrow was transplanted into lethally irradiated wild-type recipients. Consistent with the global Irf8
deficient mice, Irf8–/–
bone marrow chimeric mice showed severe high turnover osteoporosis (). IRF8 is mainly expressed in hematopoietic cells7–9
. Indeed, IRF8 expression was not detected in primary calvarial osteoblasts (data not shown). Furthermore, primary Irf8–/–
osteoblast differentiation and matrix calcification were not affected compared to wild-type cells (Supplementary Fig. 2
online). Thus, the osteoporosis resulted from enhanced osteoclastogenesis, which is a cell autonomous consequence of Irf8
deficiency in osteoclast precursors but not osteoblasts.
Figure 2 Irf8−/− mice exhibit severe osteoporosis due to enhanced osteoclast formation. (a) Radiographic analysis of the femur and tibia. Bar, 1 cm. (b) Microcomputed tomography of the femurs of 8-week-old wild-type and Irf8−/− (more ...)
On the basis of these results, we examined the differentiation potential of osteoclast precursors obtained from Irf8–/– mice. When wild-type or Irf8–/– osteoclast precursors were cocultured with wild-type or Irf8–/– primary osteoblasts in the presence of active vitamin D3 (an inducer of RANKL expression in osteoblasts), a greater number of osteoclasts formed from the Irf8–/– precursors than from the wild-type cells, independent of whether the osteoblasts were from Irf8–/– or wild-type mice (). These data further support the notion that there is no difference in osteoclastogenic activity between wild-type and Irf8–/– osteoblasts, and that Irf8 deficiency in osteoclast precursors plays a decisive role in promoting osteoclast differentiation. Indeed, macrophage cultures prepared from Irf8–/– mice also exhibited augmented osteoclastogenesis in the presence of RANKL and M-CSF (), which led to an increase in resorption on dentin slices in vitro (data not shown). On the other hand, retrovirus-mediated reconstitution of IRF8 expression in Irf8–/– macrophages inhibited RANKL-induced osteoclastogenesis (). These results demonstrate that IRF8 in osteoclast precursors is involved in the inhibition of osteoclastogenesis in vitro and in vivo. Furthermore, we found that IRF8 expression was also decreased during RANKL-induced human osteoclastogenesis, although with slower kinetics (). Silencing of Irf8 mRNA in human osteoclast precursors by siRNAs resulted in enhanced osteoclast differentiation (), indicating the function of IRF8 in osteoclastogenesis is well conserved in humans and mice.
Figure 3 IRF8 deficiency or RNAi-mediated silencing in osteoclast precursors leads to enhanced osteoclast formation. (a) Primary calvarial osteoblasts and bone marrow cells obtained from wild-type and Irf8−/− mice were cocultured in the presence (more ...)
Consistent with the results regarding osteoclastogenesis, the mRNA expression profiles of various osteoclast markers were more strongly upregulated by RANKL stimulation in macrophages prepared from Irf8–/–
mice compared with those obtained from wild-type mice (Supplementary Fig. 3
online). However, Rank
mRNA expression and RANK cell surface protein expression were comparable in wild-type and Irf8–/–
osteoclast precursors (Supplementary Fig. 4a
online and data not shown). During osteoclast precursor generation from bone marrow cells, M-CSF treatment did not lead to a greater increase in Rank
mRNA expression in Irf8–/–
cells than in control cells (Supplementary Fig. 4b
online). These results suggest that enhanced osteoclastogenesis in Irf8–/–
macrophages is not due to changes in these receptor levels.
Expression of Nfatc1 gene and the osteoclast marker Acp5 gene that encodes tartrate-resistant acid phosphatase (TRAP) protein in Irf8–/– precursors was induced by concentrations of RANKL that were 5%–25% of those required for the expression of these genes in wild-type precursors (). Furthermore, overexpression of IRF8 in the precursor cells repressed the RANKL-induced expression of Nfatc1 and Acp5 mRNAs ().
Figure 4 IRF8 inhibits NFATc1 transcriptional activity and expression, and reduced IRF8 expression may contribute to pathological bone destruction. (a) Expression of Nfatc1 and Acp5 mRNAs induced by 24 h stimulation of BMMs derived from wild-type and Irf8−/− (more ...)
These observations led us to examine the effect of IRF8 on the transcriptional activity of NFATc1, which was previously reported to interact with IRF815
. For these experiments, we employed luciferase reporter plasmids driven by three copies of the NFATc1 binding site from the human IL-2
distal promoter (p3x Nfatc1
) or by the mouse Acp5
). Overexpression of Nfatc1
gene activated these promoters, whereas simultaneous expression of Irf8
gene reduced the activities of the promoters to control levels (), indicating that IRF8 inhibits the transcriptional activity of NFATc1. When GST-IRF8 proteins were incubated with nuclear lysates containing FLAG-hemagglutinin-tagged NFATc1 (FH-NFATc1), anti-FLAG antibodies resulted in the coimmunoprecipitation of GST-IRF8 and FH-NFATc1, suggesting that IRF8 physically interacts with NFATc1 (). Furthermore, association of endogenous IRF8 and NFATc1 was identified by co-immunoprecipitation from nuclear extracts of human monocytic cells (Supplementary Fig. 5
online). We further examined the effects of IRF8 on the binding of NFATc1 to its target DNA elements in an electrophoretic mobility shift assay (EMSA); NFATc1-DNA complexes were detected, which was confirmed by the addition of competitive probes or anti-NFATc1 antibodies. Increased levels of GST-IRF8 or nuclear lysates containing excess IRF8, however, significantly decreased the binding of NFATc1 to the probes (), demonstrating the inhibitory effect of IRF8 on NFATc1 binding to its target DNA elements.
We then attempted to examine the roles of IRF8 in the processes underlying pathological bone destruction. Because several members of the IRF family of transcription factors, including IRF8, have been demonstrated to play crucial roles in toll-like receptor (TLR) signaling in response to such microbial components as LPS and unmethylated CpG DNA16,17
, we examined the role of IRF8 in the bone destruction observed during TLR-mediated inflammation. Administration of LPS to the calvarial periosteum resulted in enhanced osteoclast formation in wild-type mice, whereas more extensive bone destruction was observed in Irf8–/–
mice (). These results suggest that IRF8 is a critical negative regulator of osteoclastogenesis and a mediator of the maintenance of bone integrity during inflammatory bone destruction. We also examined the effects of TLR ligands on osteoclastogenesis in wild-type and Irf8–/–
osteoclast precursors. As previously reported18
, LPS at high doses completely inhibited RANKL-induced osteoclastogenesis in wild-type cell cultures. In contrast, corresponding high doses of LPS, and also of the TLR3 ligand poly(I:C) and the TLR9 ligand CpG DNA, only partially inhibited osteoclast differentiation in Irf8–/–
cell cultures ( and Supplementary Fig. 6
on line). Irf8
-deficient cells were almost completely refractory to the inhibitory effects of the TLR2 ligand peptidoglycan (Supplementary Fig. 6a
). These results suggest a potential inhibitory role of IRF8 in the regulation of osteoclastogenesis by TLRs, although TLRs can also activate IRF8-independent inhibitory mechanisms. We further found that Ifnα/β
expression was not diminished in Irf8
-deficient cells, and that IFN-γ completely inhibited osteoclastogenesis in Irf8–/–
macrophages, similar to wild-type cells (data not shown), thus suggesting that IRF8 can inhibit osteoclastogenesis independently of IFN-γ.
Finally, we examined the effect of TNFα on osteoclastogenesis using Irf8–/–
precursors, because TNFα is a critical mediator of inflammation induced by TLRs19–21
and has been suggested to be able to induce osteoclastogenesis22–24
. Consistent with previous reports, TNFα induced the development of a small number of osteoclasts in cultures of wild-type precursor cells (). Notably, osteoclastogenesis was enhanced in cultures of Irf8–/–
precursor cells treated with TNFα (). The mRNA expression levels of Nfatc1
in the Irf8–/–
osteoclast precursors were also augmented by TNFα (), indicating that IRF8 also plays a suppressive role in TNFα–induced osteoclastogenesis.
Our data provide a mechanism by which IRF8 suppresses osteoclastogenesis (depicted in Supplementary Fig. 7
on line). In osteoclast precursors, abundant IRF8 interacts with basally expressed NFATc1 to suppress its transcriptional activity and thus prevent its activation of target genes, including autoamplification of its own promoter. Stimulation of osteoclast precursors with RANKL results in the activation of NF-κB and AP-1 that bind to the Nfatc1
promoter to induce its activity. At the same time, RANKL induces the downregulation of IRF8, thereby releasing NFATc1 from IRF8-mediated suppression and augmenting NFATc1-mediated auto-amplification of its own expression. Together, these mechanisms result in robust NFATc1 expression and induction of downstream genes required for osteoclast differentiation.
Bone erosion that occurs in the setting of infection and chronic inflammation, termed inflammatory osteolysis, contributes to the pathogenesis of infectious and inflammatory diseases such as rheumatoid arthritis25
. Inflammatory bone erosion is driven by microbial products such as LPS and inflammatory cytokines including TNFα that activate osteoclastogenesis directly or indirectly via activation of stromal cells and osteoblasts25
. Our findings show that IRF8 plays an important role in attenuating LPS-induced inflammatory bone resorption and LPS- and TNFα-induced osteoclastogenesis. This homeostatic role of IRF8 may be important during acute infections and also in chronic inflammatory conditions such as rheumatoid arthritis. Identification of additional factors and mechanisms that augment IRF8 expression or function may represent a fruitful approach to therapeutic suppression of inflammatory bone erosion.
Overall, our findings support a model of RANKL-induced NFATc1 expression and osteoclast differentiation that involves cooperation between RANKL-mediated induction of positive regulators of osteoclastogenesis and suppression of negative regulators of osteoclast differentiation such as IRF8.