Interferon regulatory factor-8 regulates bone metabolism by suppressing osteoclastogenesis

Abstract

Bone metabolism results from a balance between osteoclast-driven bone resorption and osteoblast-mediated bone formation. Diseases such as periodontitis and rheumatoid arthritis are characterized by increased bone destruction due to enhanced osteoclastogenesis. Here we report that interferon regulatory factor-8 (IRF-8), a transcription factor expressed in immune cells, is a key regulatory molecule for osteoclastogenesis. IRF-8 expression in osteoclast precursors was downregulated during the initial phase of osteoclast differentiation induced by receptor activator of nuclear factor-kappaB ligand (RANKL), which is encoded by the Tnfsf11 gene. Mice deficient in Irf8 showed severe osteoporosis, owing to increased numbers of osteoclasts, and also showed enhanced bone destruction after lipopolysaccharide (LPS) administration. Irf8-/- osteoclast precursors underwent increased osteoclastogenesis in response to RANKL and tumor necrosis factor-alpha (TNF-alpha). IRF-8 suppressed osteoclastogenesis by inhibiting the function and expression of nuclear factor of activated T cells c1 (NFATc1). Our results show that IRF-8 inhibits osteoclast formation under physiological and pathological conditions and suggest a model where downregulation of inhibitory factors such as IRF-8 contributes to RANKL-mediated 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 analysis of IRF8 and NFATc1 expressions in cytoplasmic and nuclear fractions of BMMs obtained 0, 8, 24 and 96 h after stimulation with M-CSF and RANKL. Expression levels of TBP and p38 were measured as loading controls for nuclear and cytoplasmic fractions, respectively. (c) Inhibition of GFP-positive multinucleated cell (osteoclast) formation due to retrovirus-mediated overexpression of IRF8 in BMMs. Overexpression of IRF8 protein in BMMs was confirmed by immunoblotting (top left). GFP-positive multinucleated cells were counted (top right). GFP-positive multinucleated cells appear green giant cells (bottom). Control, pMX-IRES-EGFP; Irf8, pMX-Irf8-IRES-EGFP. **P<0.01. Bar, 50 µm. (d) BMMs were transduced with pMX-puro (control) or pMX-Irf8-puro (Irf8) and stimulated with M-CSF and RANKL for 3 days. Phagocytic activity was examined using FITC-conjugated zymosan bioparticles (green). F-actin was labeled with rhodamine-conjugated phalloidin (red). Nuclei were stained with DAPI (blue). Colocalization of zymosan and actin is denoted by the yellow fluorescent signals. Bar, 100 µm.

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^−/− mice (left), and bone morphometric analysis of femurs isolated from8-week-old mice (n = 6 in each group) (right). BV/TV, bone volume per tissue volume; Tb.Th, trabecular bone thickness; Tb.N, trabecular number; N.Nd/TV, number of nodules per tissue volume. **P<0.01; *P<0.05. Bar, 500 µm (c) Histology of femurs from 8-week-old mice in which osteoclasts were stained using TRAP, an enzymatic marker of osteoclasts (left), and histomorphometric analysis of tibias from 8-week-old mice (n=5 in each group) (right). Oc.S/BS, osteoclast surface per bone surface; N.Oc/BS, number of osteoclasts per bone surface. *P<0.05. Bar, 100 µm. (d) Histological photographs of bone formation that show tetracycline-calcein double labeling, which were administered with an interval of 72 h (left), and histomorphometric analysis of the bone formation rate and bone resorption rate in 8-week-old mice (right). BFR/BS, bone formation rate per bone surface; BRs.R, bone resorption rate. **P<0.01. (e) Analysis of IRF8 protein levels in BMs or M-CSF induced BMMs from recipient chimeric mice. (f) Soft-X ray photographs of long bones (tibias and femurs) isolated from chimeric mice. (g) Histological analysis of tibias isolated from chimeric mice (Villanueva bone staining). Bar, 500 µm. (h) Bone morphometric analysis of femurs isolated from chimeric mice. Representative data from one of two independent experiments is shown [n=3 (WT) and 3 (Irf8^−/−) in each experiment]. Data are expressed as the mean+SD (n=3). **P<0.01; *P<0.05.

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 of 10⁸ M 1,25(OH)₂D₃ and 10⁻⁶ M prostaglandin E₂ (inducers of RANKL expression in osteoblasts) for 6 days. TRAP staining (left) and TRAP activity (right) of cultures are shown. Bar, 50 µm. (b) Osteoclast formation induced by 50 ng/ml M-CSF and the indicated doses of RANKL in BMM cultures. TRAP staining (left) and TRAP activity (right) of cultures are shown. Bar, 50 µm. (c) Irf8^−/− macrophages were transduced with the vectors such as pMX-Irf8-IRES-EGFP (Irf8) or pMX-IRES-EGFP (Control) and stimulated with 150 ng/ml RANKL for 3 days (left). GFP-positive multinucleated cells were counted as osteoclasts (right). **P<0.01. Bar, 50 µm. (d) Kinetics of Irf8 mRNA expression during human osteoclastogenesis induced by 40 ng/ml RANKL at indicated time points. (e) Human CD14-positive monocytic cells were transfected with human Irf8-specific short interfering RNAs (si-hIrf8) or non-targeting control siRNAs (si-control), cultured for 2 days in the presence of 20 ng/ml M-CSF, and efficiency of silencing of Irf8 mRNA was examined by quantitative real time-PCR. (f) The cells were further stimulated with indicated concentrations of RANKL for 6 days. Number of TRAP-positive multinucleated cells was counted as osteoclasts (left). TRAP-positive cells appear red in the photograph (right). **P<0.01. Representative data from one of three donors is shown; similar results were obtained using a distinct Irf8-specific siRNA in an additional three experiments. Data are expressed as the mean+SD of quad-duplicate cultures. **P<0.01; n.s., no statistical difference. Bar, 50 µm.

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^−/− mice with 50 ng/ml M-CSF and the indicated doses of RANKL (Northern blotting). (b) Expression of Nfatc1 and Acp5 mRNAs in retrovirus-infected BMMs (Control, pMX-puro; Irf8, pMX-Irf8-puro) in the absence or presence of 150 ng/ml RANKL with 50 ng/ml M-CSF for 24 h (Northern blotting). Gapdh was used as an internal control. (c) A luciferase activity assay to examine the effect of IRF8 on the transcriptional activity of NFATc1. **P<0.01. (d) The interaction between NFATc1 and IRF8. IP, immunoprecipitation; IB, immunoblotting. (e,f) Analysis of the inhibition of NFATc1 binding to its target DNA sequences using EMSAs (see supplementary methods on line). The arrow heads indicate a specific binding complex, which included the NFATc1–DNA complex; this was confirmed in a supershift assay (lane 1–3) and a cold competition experiment (lane 4). The intensity of NFATc1–DNA bands decreased with increasing amounts of GST-IRF8 (lanes 7–9), or when nuclear extracts from HEK293 cells cotransfected with pcDNA3-Nfatc1 and pcDNA3-Irf8 were used (f), but not with GST alone (lanes 5–6). (g) TRAP staining of mouse whole calvaria (left) and histological sections (right) obtained from wild-type and Irf8^−/− mice with or without administration of LPS. (h) Osteoclast formation in BMMs stimulated with various doses of LPS in the presence of M-CSF (50 ng/ml) and RANKL (150 ng/ml) for 5 days. Data are expressed as the mean+SD of 4 cultures. (i) Left, BMM cultures in the presence of 50 ng/ml M-CSF and 0 or 25 ng/ml TNFα on day 3 shown by TRAP staining. Right, TNFα dose-dependent induction of osteoclast formation examined using TRAP activity assays. (j) Expression of Nfatc1 and Acp5 mRNAs in BMMs derived from wild-type and Irf8^−/− mice stimulated with 50 ng/ml M-CSF and the indicated doses of TNFα for 24 h (Northern blotting).