The transcription factor FBI-1/OCZF/LRF is expressed in osteoclasts and regulates RANKL-induced osteoclast formation in vitro and in vivo

Authors


Abstract

Objective

Since transcription factors expressed in osteoclasts are possible targets for regulation of bone destruction in bone disorders, we investigated the expression of the transcription factor FBI-1/OCZF/LRF (in humans, factor that binds to inducer of short transcripts of human immunodeficiency virus type 1; in rats, osteoclast-derived zinc finger; in mice, leukemia/lymphoma-related factor) in patients with rheumatoid arthritis (RA), and assessed its role in osteoclastogenesis in vivo.

Methods

Expression of FBI-1/OCZF was investigated in subchondral osteoclasts in human RA and in rat adjuvant-induced arthritis (AIA) using immunostaining and in situ hybridization, respectively. Transgenic mice overexpressing OCZF (OCZF-Tg) under the control of the cathepsin K promoter were generated, and bone mineral density and bone histomorphometric features were determined by peripheral quantitative computed tomography, calcein double-labeling, and specific staining for osteoclasts and osteoblasts. LRF/OCZF expression and the consequence of LRF inhibition were assessed in vitro with RANKL-induced osteoclast differentiation.

Results

FBI-1/OCZF was detected in the nuclei of osteoclasts in rat AIA and human RA. RANKL increased the levels of LRF messenger RNA and nuclear-localized LRF protein in primary macrophages. In OCZF-Tg mice, bone volume was significantly decreased, the number of osteoclasts, but not osteoblasts, was increased in long bones, and osteoclast survival was promoted. Conversely, inhibition of LRF expression suppressed the formation of osteoclasts from macrophages in vitro.

Conclusion

FBI-1/OCZF/LRF regulates osteoclast formation and apoptosis in vivo, and may become a useful marker and target in treating disorders leading to reduced bone density, including chronic arthritis.

Bone destruction in rheumatoid arthritis (RA) depends on the formation of osteoclasts in the inflamed joints. Osteoclasts, the principal bone-resorbing multinucleated cells, are derived from cells of the monocyte/macrophage lineage. Recent advances in elucidating the mechanism of osteoclast differentiation revealed that RANK and its ligand, RANKL, which is a member of the tumor necrosis factor (TNF) superfamily, are critical molecules in osteoclastogenesis from hematopoietic stem cells (1, 2). RANKL also has a central role in inflammatory bone loss and is expressed in activated T cells and fibroblast-like synovial cells in RA, inducing osteoclastogenesis (3, 4).

In downstream signaling through RANK, RANKL activates transcription factors, such as NF-κB, which is a common signaling molecule of the TNF family, as well as c-Fos and nuclear factor of activated T cells c1 (NF-ATc1) (5, 6). Studies in knockout mice have demonstrated that these transcription factors are essential for osteoclastogenesis in vivo (7–9). The antirheumatic drug leflunomide inhibits osteoclastogenesis by interfering with the induction of NF-ATc1 (10). Thus, regulation of transcription factors in RANK signaling is necessary to control excessive bone destruction.

FBI-1/OCZF/LRF (in humans, factor that binds to inducer of short transcripts of human immunodeficiency virus type 1; in rats, osteoclast-derived zinc finger; in mice, leukemia/lymphoma-related factor) belongs to the POK (BTB/POZ and Krüppel) family of transcription factors and has 2 conserved domains, the N-terminal BTB/POZ domain and the C-terminal zinc fingers, which are involved in cell differentiation and oncogenesis (11–14). The BTB/POZ domain contributes to transcriptional repression by interacting with transcriptional corepressor complexes (15). OCZF is a rat member of the POK family, and its human and mouse orthologs are known as FBI-1 and LRF, respectively (16–18). This transcription factor is encoded by the Zbtb7a gene and interacts with BCL-6, which is involved in B cell lymphoma and inflammation (12). Knockout studies have shown that LRF is a critical regulator of T and B lymphopoiesis (19). Recently, it was reported that LRF has an antiapoptotic role in erythroid differentiation, suggesting that it plays a role in terminal differentiation of these cells (13).

OCZF was originally cloned by immunoscreening a complementary DNA (cDNA) library, which was constructed from a rat cell population enriched in osteoclasts with the osteoclast-specific monoclonal antibody Kat6 (16, 20). We have shown that OCZF is preferentially expressed in rat mandibular osteoclasts (16). However, it is not known whether FBI-1 is involved in pathologic osteoclastogenesis in humans, and whether OCZF/LRF has a role in RANKL-induced osteoclastogenesis in vivo. In the present study, we analyzed the expression of FBI-1/OCZF in subchondral osteoclasts in human RA and in rat adjuvant-induced arthritis (AIA). Furthermore, we generated transgenic mice overexpressing OCZF (OCZF-Tg) under the control of the cathepsin K promoter. Our results indicate that FBI-1/OCZF/LRF is highly expressed in osteoclasts and is an important regulator of RANKL-induced osteoclastogenesis and the associated loss of bone mass.

MATERIALS AND METHODS

Mice.

Male DDY mice (ages 5–7 weeks) and female Lewis rats (age 5 weeks) were obtained from Seac Yoshitomi. Experiments were carried out according to the protocol approved by the Laboratory Animal Care and Use Committee of Saga University and Kyusyu University.

Antibodies.

Antibodies against NF-ATc1 (sc-7294) and actin (sc-1615) were purchased from Santa Cruz Biotechnology. To prepare the anti-OCZF polyclonal antibodies, an OCZF glutathione S-transferase (GST) fusion gene was prepared by inserting the Bam HI/Sma I fragment of OCZF cDNA (amino acids 12–292) into the pGEX vector (GE Healthcare). Rabbit anti-OCZF polyclonal IgG was then produced by immunizing the animals with purified recombinant OCZF-GST fusion protein.

Constructs.

The mouse cathepsin K (Ctsk) promoter fragment (−2057 to −48 bp), which has homology to the corresponding region in the human Ctsk promoter, was amplified by polymerase chain reaction (PCR) from mouse genomic DNA using the primers 5′-GGGGTACCCCTTTACAGACATGCACAACT-3′ and 5′-GAAGATCTGGCTACTGTGAGCGGAAGAC-3′ (21). The fragment was then cloned into the pGL3-basic luciferase vector (Promega) and designated pGL3-Ctsk-luc. Reporter plasmids harboring −1,676 to −48 bp (1.6 kb) and −666 to −48 bp (0.6 kb) were constructed using restriction sites of Xba I and Pma CI, respectively. To prepare plasmid pGL3-Ctsk-OCZF, the coding region (1,710 bp from start to stop codons) of OCZF cDNA was cloned into Xho I– and Bam HI–cut pIRES2-EGFP (Clontech). The fragment including OCZF and EGFP cDNA (2,015 bp in length) was then excised by Nhe I–Not I digestion and cloned into Nhe I– and Xba I–cut pGL3-Ctsk-luc, which contains the Ctsk promoter up to −1676 bp.

Immunohistochemical analysis of human tissue.

The spatial expression of FBI-1 protein was determined immunohistochemically, using joint tissue obtained at the time of arthroplasty from a patient with RA who fulfilled the American College of Rheumatology criteria for the diagnosis of RA (22). Five-micrometer–thick sections were cut on a rotatory microtome, from a formalin-fixed, paraffin-embedded specimen. Tissue integrity and the histopathologic appearance of the specimen were assessed in a hematoxylin and eosin (H&E)–stained section. An automated staining system (Autostainer Plus; Dako) was used for immunostaining, using hamster anti–FBI-1/LRF monoclonal IgG (23) at a dilution of 1:5,000, followed by overnight incubation at 4°C. Antigen retrieval (at 100°C for 5 minutes in 0.01M citrate buffer, pH 7.6) was used.

Generation of rat AIA and in situ hybridization for detection of OCZF messenger RNA (mRNA).

AIA was induced in rats by a single intradermal injection (into the tail base) of heat-killed Mycobacterium butyricum (25 mg/kg) suspended in mineral oil. Paw erythema and swelling were noted 10 days later, and both symptoms increased in severity over the following 2 weeks. Rats were anesthetized with pentobarbital at 21 days after adjuvant injection and were perfused with 4% paraformaldehyde in phosphate buffered saline (PBS). Hind paw tissue containing the tibia was immersed in the same fixative, demineralized in Morse's solution for 3 weeks, and then embedded in paraffin.

Sense and antisense RNA probes corresponding to the 5′-terminal region of the OCZF mRNA (158–785) were labeled with digoxigenin (DIG). In situ hybridization was performed using a DIG nucleic acid detection kit (Roche), as described previously (16).

Osteoclast formation and survival assay in vitro.

The murine macrophage cell line RAW-D (a subclone of RAW264.7) has a high potential to differentiate into osteoclasts (24). RAW-D cells were cultured in α-minimum essential medium containing 10% fetal calf serum. For differentiation, RAW-D cells were cultured for 3–4 days at a density of 4.5 × 104 cells/ml in the presence of 20 ng/ml soluble RANKL (PeproTech) and 5 ng/ml TNFα (Roche). To induce formation of rat osteoclasts, bone marrow cells were prepared from the femurs of male rats, and cultured in the presence of 10−8M 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) as previously described (25). To induce formation of mouse osteoclasts, bone marrow cells were cultured in the presence of 10 ng/ml macrophage colony-stimulating factor (M-CSF) for 3 days to generate bone marrow macrophages, which were then stimulated with 50 ng/ml RANKL and 10 ng/ml M-CSF for an additional 2–3 days (9).

At the end of the culture, cells were fixed and stained with a commercial kit for the osteoclast marker tartrate-resistant acid phosphatase (TRAP) (Sigma-Aldrich). TRAP-positive cells with 3 or more nuclei were counted as multinucleated cells. Assays of osteoclast survival were carried out as reported previously (26). Briefly, osteoclasts were incubated for 12 hours after cytokine withdrawal. The osteoclast survival rate was then determined as the percentage of morphologically intact TRAP-positive multinucleated cells.

Transfections.

FuGene (Roche) or the Nucleofector kit T (Amaxa Biosystems) were used for transfecting RAW-D cells with plasmid DNA. Transfected cells were cultured in the presence or absence of RANKL for 1–4 days. For transfection of RAW-D cells with small interfering RNA (siRNA), the Nucleofector kit T was used. The siRNA against LRF (siRNA #1 and siRNA #2) and control siRNA against enhanced green fluorescent protein (EGFP) (iGene Therapeutics) were designed and prepared by in vitro transcription. The following primers were used: for LRF siRNA #1, 5′-CGCACAACUACGACCUGAAGAACCAAG-3′ and 5′-UGGUUGUUCAGGUCGUAGUUGUGCGAU-3′; for LRF siRNA #2, 5′-AGAAGCACUUUAAGGACGAGGAGGAAG-3′ and 5′-UCCUCCUCGUCCUUAAAGUGCUUCUAU-3′; and for EGFP siRNA, 5′-AGAACGGCAUCAAGGUGAACUUCAAAG-3′ and 5′-UUGAAGUUCACCUUGAUGCCGUUCUAU-3′.

For luciferase assays, the cells were harvested in Promega Lysis Buffer 48 hours after transfection. Luciferase levels were measured using a Dual Luciferase kit (Promega), and luciferase activity was normalized against the activity of the Renilla luciferase plasmid pΔTK-RL, which contains the basal thymidine kinase promoter,

Immunofluorescence.

Cells were stained as described previously (27). Briefly, cells were fixed in 99:3 cold methanol:formaldehyde for 20 minutes at −20°C, permeabilized in 0.2% Triton X-100 for 10 minutes at room temperature, and then blocked by incubation in 3% normal goat serum for 60 minutes at room temperature. The cells were then incubated with rabbit anti-OCZF antibodies for 60 minutes. The cells were washed and then incubated with secondary antibodies (goat anti-rabbit Alexa Fluor 488) obtained from Invitrogen. The cells were examined with an LSM 5 Pascal confocal laser scanning microscope (Carl Zeiss).

Western blot analysis.

For isolation of cell extracts, the cells were washed with ice-cold PBS and suspended in sodium dodecyl sulfate (SDS) buffer (0.125M Tris HCl, pH 6.8, 2% SDS, 10% glycerol). Proteins were fractionated with 8–10% polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The filters were washed with Tris buffered saline–0.05% Tween 20 (TBST), blocked in TBST containing 5% low-fat milk at room temperature for 1 hour, and probed with antibodies for 1 hour. The filters were then incubated with secondary antibodies.

The antibody detection reaction was performed with an enhanced chemiluminescence Western blotting system (GE Healthcare). Relative protein expression was calculated from band densities using an LAS-3000 image analyzer (Fujifilm) and Multi Gauge image analysis software (Fujifilm).

Real-time quantitative reverse transcription–PCR (RT-PCR).

Total RNA was extracted from the mouse cells using a commercial kit (Isogen; Nippon Gene), and digested with DNase to eliminate genomic DNA. The cDNA was synthesized and assayed on an ABI 7900HT sequence detector (Applied Biosystems) for relative expression of LRF and GAPDH. Relative mRNA levels were determined by the comparative threshold method (28), and results were normalized to the levels of GAPDH mRNA. Commercially available probe/primer sets (Applied Biosystems) with proprietary sequences were used. The sizes of the expected amplification products are publically available and were verified by electrophoretically resolving the PCR products on 2% agarose gels.

Generation of OCZF-Tg mice.

A 1.6-kb fragment that includes the Ctsk promoter, OCZF, and EGFP cDNA was excised from pGL3-Ctsk-OCZF and injected into pronuclei of fertilized BDF-1 eggs, as described previously (29). Production of OCZF-Tg mice resulted in 66 pups, among which 20 founder mice were produced. The genotypes of the mice were analyzed either by Southern blotting, using full-length OCZF cDNA as a probe, or by PCR analysis. OCZF transgene–negative littermates were used as wild-type (WT) controls. All transgenic mice were viable and reproduced normally. Transgenic mouse lines were maintained on a C57BL/6 background.

Bone and histologic analyses.

For bone analysis, the right femurs of 6-week-old male mice were dissected and stored in 70% ethanol for peripheral quantitative computed tomography (pQCT), microfocal CT (micro-CT) scanning, or radiography analysis. In pQCT analysis, bones from 6-week-old WT or OCZF-Tg mice (n = 7–10 per genotype) were measured using an XCT Research SA+ scanner (Stratec Medizintechnik). The images were scanned with a voxel size of 0.08 × 0.08 × 0.46 mm3. Trabecular and cortical parameters were obtained 1.2 mm from the distal growth plates and at the mid point of the diaphysis, respectively. Threshold values of 690 mg/cm3 for the cortical region and 395 mg/cm3 for the trabecular region were used, as described previously (30).

Micro-CT scanning was performed on the distal metaphyses of the femurs using a micro-CT Scan Xmate-A0905. The left femurs were fixed in 4% paraformaldehyde and then decalcified with EDTA. Paraffin-embedded sections were stained with H&E. GFP expressed in bone tissues was detected by an anti-GFP antibody conjugated with Alexa 488 (Invitrogen).

For bone histomorphometry, 6-week-old male mice were injected twice with calcein (1 and 3 days before the mice were killed). Long bones were fixed in 4% paraformaldehyde and undecalcified bones were embedded in Technovit 7100, according to the manufacturer's protocol. Sections from the metaphyses of the tibiae were cut longitudinally in the proximal region and stained with toluidine blue. Histomorphometric measurements were performed in cancellous bone in the methaphyses of the proximal tibiae.

To determine the numbers of osteoclasts and osteoblasts, sections were stained for TRAP and alkaline phosphatase activity, respectively. Units used are those recommended by the Nomenclature Committee of the American Society for Bone and Mineral Research (31).

Statistical analysis.

Statistical significance was determined with Student's t-tests. P values less than 0.05 were considered significant.

RESULTS

High expression of OCZF and FBI-1 in osteoclasts in rat AIA and human RA.

We first analyzed the expression of OCZF mRNA in osteoclasts of rats with AIA. OCZF mRNA in the rat AIA model was detected extensively in multinucleated osteoclasts in the distal tibiae of arthritic rat hind paws displaying severe bone destruction (Figure 1A, arrows). In the same specimen, strong staining was also observed in marrow mononuclear cells in close association with bone (Figure 1B). In both cases, no signal was seen when the sense probes were used as negative controls.

Figure 1.

Expression of transcription factor FBI-1/OCZF (in humans, factor that binds to inducer of short transcripts of human immunodeficiency virus type 1; in rats, osteoclast-derived zinc finger) in subchondral osteoclasts in rat adjuvant-induced arthritis (AIA) and human rheumatoid arthritis (RA). A and B, OCZF mRNA was detected by in situ hybridization in bone marrow (BM) from the distal tibia of an affected hind limb of a rat with AIA. The antisense RNA probe detected OCZF mRNA in the cytoplasm (arrows) of multinucleated osteoclasts (A) and also in large mononuclear cells, the presumed precursors to osteoclasts, in close approximation to bone (Bo) (B). OCZF mRNA was not detected with a sense RNA probe as control. Antisense and sense strand RNA probes, corresponding to the 5′-terminal region of OCZF mRNA (158–785), were used. C, In a joint tissue specimen obtained during arthroplasty from a patient with end-stage RA, FBI-1+ nuclear staining was found in a multinucleated lacunar osteoclast (arrow). Original magnification × 630.

To examine FBI-1 protein expression in human osteoclasts, we analyzed the expression of FBI-1 using joint tissue from a patient with late-stage RA. Strong nuclear FBI-1 staining was evident in multinucleated lacunar osteoclasts in the subchondral marrow space of the RA joint tissue, as shown in Figure 1C (arrow). These findings indicate that FBI-1/OCZF is specifically and highly expressed in inflammatory arthritis in osteoclasts and preosteoclasts in subchondral bone.

RANKL-stimulated increase in expression of OCZF/LRF during osteoclast differentiation in vitro.

To examine the induction of OCZF/LRF expression in osteoclasts formed in bone marrow culture in the presence of RANKL, immunostaining with anti-OCZF antibodies was performed. As shown in Figure 2A, the accumulation of OCZF in multinucleated and mononuclear cells, which clustered near stromal cells after the addition of 1,25(OH)2D3, was detected in rat bone marrow culture.

Figure 2.

Effects of RANKL on the expression of rat osteoclast-derived zinc finger (OCZF)/mouse leukemia/lymphoma-related factor (LRF) during osteoclastogenesis in vitro. A, Rat bone marrow cells were cultured in the presence of 10−8M 1,25-dihydroxyvitamin D3 for 4 days, and OCZF was detected in rat osteoclasts by immunofluorescence using anti-OCZF antibodies (right); a phase contrast image from fluorescence microscopy is also shown (left). B, Relative LRF mRNA levels were determined in mouse bone marrow macrophages by real-time polymerase chain reaction after the cells had been cultured with macrophage colony-stimulating factor (M-CSF) (10 ng/ml) and RANKL (50 ng/ml) for the indicated number of days. Values were normalized to those for GAPDH. C, Western blotting was used to assess the expression of LRF and nuclear factor of activated T cells c1 (NF-ATc1) in mouse bone marrow macrophages. Values were normalized to those for actin. Values below the blots are the relative intensity of the bands. D, Immunofluorescence analysis confirms the presence of LRF in mouse bone marrow macrophages cultivated with or without M-CSF and RANKL (top). Differential interference contrast (DIC) images are also shown (bottom). Bars = 50 μm. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

When mouse bone marrow macrophages were treated with RANKL and M-CSF, multinucleated osteoclasts formed efficiently. LRF mRNA was detected in mouse bone marrow macrophages, and the levels increased after stimulation with RANKL, reaching peak levels in ∼2 days (Figure 2B). We then analyzed LRF protein levels by Western blotting using the anti-OCZF antibodies. LRF protein levels also increased for 1–3 days after the addition of RANKL (Figure 2C). The levels of NF-ATc1 protein increased after the rise in LRF (Figure 2C).

Using confocal fluorescence microscopy, we investigated the subcellular localization of LRF in bone marrow macrophages treated with or without RANKL and M-CSF. An LRF signal was detected in the nuclei of multinucleated cells after stimulation with RANKL (Figure 2D). These findings indicate that RANKL specifically increases nuclear-localized LRF protein.

Decreased bone mass in OCZF-Tg mice.

To determine the physiologic role of OCZF in bone metabolism in vivo, we generated OCZF-Tg mice expressing OCZF cDNA. To this end, we cloned the Ctsk promoter to overexpress OCZF in osteoclasts. Using serially deleted Ctsk promoter–driven luciferase reporters, we found that the −1676 to −1382 region of the Ctsk promoter conferred high transcriptional activity in RAW-D cells (results not shown).

RANKL treatment markedly increased the luciferase activity driven by the −1676 Ctsk promoter (results available from the corresponding author upon request). We therefore constructed a plasmid that expresses OCZF under the control of the −1676 Ctsk promoter, and used it to generate OCZF-Tg mice. RT-PCR analysis showed that RANKL strongly increased the expression of the OCZF transgene in bone marrow macrophages derived from the OCZF-Tg mouse line (Figure 3A). Expression levels of the OCZF transgene in various tissues were correlated with those of cathepsin K, which is specifically expressed in bone tissues (results not shown). Immunohistochemical analysis with an anti-GFP antibody showed that GFP was mostly expressed in TRAP-positive cells of OCZF-Tg mice, although some GFP-positive cells were not TRAP-positive (Figure 3B).

Figure 3.

Bone mass is decreased in transgenic (Tg) mice expressing osteoclast-derived zinc finger (OCZF) under the control of the cathepsin K promoter, in comparison with wild-type (WT) mice. A, Reverse transcription–polymerase chain reaction was used to analyze the transgenic expression of OCZF and expression of Ctsk in bone marrow macrophages derived from WT and OCZF-Tg mice, after the cells had been cultivated with or without RANKL for 24 hours. B, Bone tissue from WT and OCZF-Tg mice was analyzed immunohistochemically with an anti–green fluorescent protein (anti-GFP) antibody (Ab) and staining for the osteoclast marker tartrate-resistant acid phosphatase (TRAP). Bar = 20 μm. C, Femurs from 6-week-old WT and OCZF-Tg mice were assessed for bone mass changes by hematoxylin and eosin (H&E) staining, radiography, and microfocal computed tomography (μCT; micro-CT). Micro-CT images were scanned at a distance equal to one-tenth of the total length of the femur from the distal growth plate. Representative samples are shown. Bar = 2 mm. D, Femurs from WT and OCZF-Tg mice were assessed by peripheral quantitative computed tomography analysis to measure the bone mineral density (BMD) and bone mineral content (BMC) in total, trabecular, and cortical bones. The stress strain index (SSI) of trabecular bones was also measured. Horizontal lines indicate the mean for each group. ∗ = P < 0.05; ∗∗ = P < 0.005; # = P < 0.0005 versus WT, by Student's t-test. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

In studies with animals at 6 weeks of age, we analyzed the histologic appearance of H&E-stained serial sections of the distal femurs from male and female OCZF-Tg mice, along with sections from femurs from WT mice. As evident in the representative H&E-stained sections from female mice at 6 weeks of age (shown in Figure 3C, top), trabecular bone was decreased in the metaphyses of the distal femurs from OCZF-Tg mice in comparison with WT mice. In addition, comparison of the images from radiography and micro-CT analyses of the femurs of WT and OCZF-Tg mice similarly revealed that the amount of trabecular bone was reduced in OCZF-Tg mice (Figure 3C, middle and bottom).

We also compared bone mineral density (BMD) and bone mineral content (BMC), as determined by pQCT analysis, in the total, trabecular, and cortical regions of the distal femurs between 6-week-old OCZF-Tg mice and WT mice. As shown in Figure 3D, BMC and BMD were significantly decreased (by 40% and 36%, respectively) in trabecular bone of OCZF-Tg mice. The stress strain index, an index of bone strength, was also decreased, by 29%, in OCZF-Tg mice compared with WT mice (Figure 3D). Thus, the histologic appearance of the distal femurs of 6-week-old OCZF-Tg mice indicated that osteopenia had developed in these mice.

Increased formation of osteoclasts, but not osteoblasts, in OCZF-Tg mice.

To determine whether the decrease in trabecular bone density was due to increased bone resorption or to decreased bone formation, bone histomorphometric analysis was performed in OCZF-Tg and WT mice. We analyzed the number of TRAP-positive osteoclasts in the metaphyses of the proximal tibiae of 6-week-old male mice. The numbers of osteoclasts and percentage of osteoclast surfaces in the trabecular bones of OCZF-Tg mice were significantly increased (by 224% and 189%, respectively) when compared with that in WT mice (Figures 4A and B). In contrast, the percentage of the osteoblast surface and the rate of bone formation did not differ significantly between WT and OCZF-Tg mice (Figure 4B). These observations indicate that overexpression of OCZF in mice increased osteoclast differentiation, thereby decreasing bone mass, but did not affect bone formation.

Figure 4.

The number of osteoclasts, but not osteoblasts, is increased in transgenic (Tg) mice expressing osteoclast-derived zinc finger (OCZF) under the control of the cathepsin K promoter, when compared with wild-type (WT) mice. A, The distal femurs of WT and OCZF-Tg mice were stained for tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP), and the proximal tibiae were stained for calcein. Bar for TRAP = 400 μm; bar for ALP = 200 μm. B, Sections of the femurs from WT and OCZF-Tg mice were assessed by bone histomorphometry. Measurements were obtained in cancellous bone tissue in the methaphyses of the proximal tibiae, to determine the percentage of osteoclast (Oc.) surface, number of osteoclasts, percentage of osteoblast (Ob.) surface, and mineral apposition rate (MAR). C and D, Bone marrow macrophages from WT and OCZF-Tg mice were prepared and treated with soluble RANKL (sRANKL) and macrophage colony-stimulating factor (M-CSF) or M-CSF alone for 3 days. At the end of the culture period, cells were stained for TRAP activity, and the number of TRAP-positive multinucleated cells (MNCs) was counted (C). In addition, total cell extracts were prepared and analyzed for the expression of nuclear factor of activated T cells c1 (NF-ATc1) and c-Fos protein by Western blotting (D); actin was used as a positive control. Bars show the mean ± SEM results in 7 mice per group from 3 independent experiments. ∗ = P < 0.05 versus WT, by Student's t-test. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

To further confirm that osteoclast differentiation was increased in OCZF-Tg mice, the ability of bone marrow macrophages derived from WT and OCZF-Tg mice to undergo osteoclastogenesis was examined. As shown in Figure 4C, the number of osteoclasts was increased in OCZF-Tg mice in comparison with WT mice. In addition, Western blot analysis showed that the levels of c-Fos and NF-ATc1 in bone marrow macrophages stimulated with RANKL were also increased in OCZF-Tg mice in comparison with WT mice (Figure 4D). These findings indicate that OCZF exerted marked effects on osteoclastogenesis in vivo in the presence of RANKL, by directly affecting cells of the osteoclast lineage.

Effect of OCZF overexpression on early and late stages of osteoclastogenesis.

To study the role of OCZF in osteoclastogenesis in greater detail, we then examined the effect of OCZF overexpression on osteoclast formation from RAW-D cells in the presence or absence of RANKL. Transfection of RAW-D cells with OCZF markedly stimulated RANKL-induced osteoclast formation (Figure 5A).

Figure 5.

Overexpression of osteoclast-derived zinc finger (OCZF) affects osteoclastogenesis, expression of cell cycle–related genes, and survival of osteoclasts, but not osteoclast precursor numbers. A, RAW-D cells were transfected with control (Co) or OCZF (Oc) vector and cultured in the presence or absence of RANKL and tumor necrosis factor α for 3 or 4 days, and the number of tartrate-resistant acid phosphatase (TRAP)–positive multinucleated cells (MNCs) was assessed. B, RAW-D cells were transfected with control or OCZF vector for 2 or 4 days, and the relative expression levels of Cdk2, Cdk4, and Cdk6 (left) and Cdkn1a (p21) and Cdkn1b (p27) (right) in the presence or absence of RANKL were assessed. C, Bone marrow cells of WT and OCZF-Tg mice were analyzed for c-fms expression by fluorescence-activated cell sorter analysis (left) and for the proportion of c-fms–positive cells (right). D, The survival rate of osteoclasts was determined in WT and OCZF-Tg mice before (0 hours) and 12 hours after cytokine withdrawal. Bars show the mean ± SEM results from 3 independent experiments. ∗ = P < 0.05; ∗∗ = P < 0.01 versus control vector (in A and B) or WT (in D), by Student's t-test.

We next examined the effect of OCZF on the expression of cell cycle–related genes. During osteoclastogenesis, RANKL is known to decrease the expression of Cdk2, Cdk4, and Cdk6, but strongly increase that of the Cdk inhibitors p27KIP1 and p21CIP1 in bone marrow macrophages (32–34). In RAW-D cells, osteoclasts formed in 3 days, but not in 2 days, after the addition of RANKL.

We thus analyzed the expression of cell cycle–related genes by RT-PCR at 2 days and 4 days after RANKL stimulation (details of the methods used are available from the corresponding author upon request). OCZF inhibited p21CIP1 expression at 2 days, but increased p27KIP1 and p21CIP1 expression at 4 days, in comparison with cells transfected with control vector (Figure 5B).

We next analyzed the proportion of c-fms–positive cells, which are considered to be markers of osteoclast precursors, in bone marrow cells of WT and OCZF-Tg mice. As shown in Figure 5C, the proportion of precursor cells did not differ between the mouse genotypes. We also analyzed the survival of WT and OCZF-Tg osteoclasts. Overexpression of OCZF prevented the death of osteoclasts in response to cytokine withdrawal, whereas half of the WT osteoclasts died after 12 hours (Figure 5D).

Inhibition of osteoclast differentiation by siRNA against LRF.

To investigate the stimulatory role of LRF/OCZF in RANKL-induced osteoclastogenesis, we then analyzed the effect of knocking down LRF expression with siRNA in an in vitro culture system in which osteoclasts form from RAW-D cells. Two LRF siRNA (#1 and #2) repressed the expression of LRF mRNA and protein, but not that of β-actin protein (Figure 6A). Both of the LRF siRNA, but not the control siRNA, strongly inhibited the formation of TRAP-positive multinucleated cells from RAW-D cells (Figure 6B). In contrast, the same concentration of control siRNA did not affect osteoclast formation.

Figure 6.

Small interfering RNAs (siRNA) against leukemia/lymphoma-related factor (LRF) inhibit formation of tartrate-resistant acid phosphatase (TRAP)–positive multinucleated cells (MNCs), but not mononuclear cells, from RAW-D cells. A, Quantitative real-time polymerase chain reaction (left) and Western blotting (right) were used to assess LRF mRNA and protein expression, using total RNA or protein extracted from RAW-D cells transfected with control or LRF siRNA (#1 and #2). B–D, RAW-D cells were transfected with control or LRF siRNA and cultured in the presence of RANKL (20 ng/ml) and tumor necrosis factor α (5 ng/ml). After 3 days in culture, cells were stained for TRAP activity, and TRAP-positive MNCs (B) or mononuclear cells (D) were counted. Representative photographs of RAW-D cells transfected with control or LRF siRNA show that higher concentrations of LRF siRNA did not affect the total number of cells, but specifically inhibited the formation of TRAP-positive MNCs (C). Bars show the mean ± SEM results from 3 independent experiments. ∗ = P < 0.05; ∗∗ = P < 0.01 versus 0 μM, by Student's t-test. Bar = 50 μm.

Moreover, as shown in Figure 6C, in cultures treated with various concentrations of control and LRF siRNA, high concentrations of LRF siRNA did not affect the total number of cells, but specifically inhibited the formation of TRAP-positive multinucleated cells. Interestingly, we found that LRF siRNA did not inhibit the formation of TRAP-positive mononuclear cells (Figure 6D). These findings indicate that LRF has a role in the late phase of osteoclastogenesis.

DISCUSSION

In this study, we have shown that the transcriptional regulator FBI-1/OCZF/LRF is highly expressed in subchondral osteoclasts from patients with RA and in rat AIA, and plays a role in regulating bone resorption by increasing osteoclast formation and function in vitro and in vivo. These results suggest that FBI-1/OCZF/LRF has a role in inflammatory bone erosion and may represent a therapeutic target for treating bone loss.

To study the role of OCZF in bone resorption of osteoclasts, we chose the Ctsk promoter to overexpress OCZF in murine osteoclasts and their precursors. Cathepsin K is a lysosomal cysteine protease that degrades type I collagen and is involved in the resorptive activity of osteoclasts. It is expressed in osteoclasts and is induced by RANKL (21, 35). Bone of OCZF-Tg mice is fragile, and both BMC and BMD were significantly lower in OCZF-Tg mice than in WT controls. These differences were accompanied by a marked increase in the number of osteoclasts, but not osteoblasts. In addition, OCZF overexpression increased the level of NF-ATc1, which is a master regulator of osteoclastogenesis in bone marrow macrophages treated with RANKL in transgenic mice. These findings suggest that FBI-1/OCZF/LRF promotes osteoclastogenesis by affecting RANK signaling molecules, thereby stimulating bone resorption.

Osteoclast formation is a crucial step in bone destruction in chronic arthritis. RANKL is a critical factor for control of normal bone metabolism, but also for regulation of inflammatory bone destruction. In RA, synovial tissue provides a source of RANKL. Synovial fibroblasts express membrane-bound RANKL that induces the differentiation of synovial macrophages into osteoclasts (36). We found that the expression of LRF was markedly increased by RANKL in primary bone marrow macrophages. In vivo, strong expression of OCZF and FBI-1 protein was detected in the nuclei of subchondral osteoclasts of rats with AIA and in a patient with RA. These results suggest that the expression of FBI-1/OCZF/LRF in osteoclasts may be induced by RANKL, which originates from chronically inflamed synovium.

Differentiation of osteoclasts includes several steps, such as formation of mononuclear preosteoclasts and the fusion of preosteoclasts to form multinucleated osteoclasts. Interestingly, inhibition of LRF expression by siRNA suppressed the formation of TRAP-positive multinucleated cells, but not mononuclear cells, from RAW-D cells induced by RANKL. We also found that the expression of FBI-1 or OCZF in osteoclasts was very high compared with that of other cells in bone. Furthermore, osteoclast survival was enhanced in OCZF-Tg mice. Taken together, these results suggest that FBI-1/OCZF/LRF plays a role in the late phase of osteoclastogenesis. In contrast, analysis of the proportion of c-fms–positive osteoclast precursor cells indicated that OCZF overexpression does not affect the early phase of osteoclastogenesis.

FBI-1/OCZF/LRF is also implicated in adipogenesis (37, 38). Overexpression of FBI-1 in preadipocytes increased lipid accumulation during adipogenesis and reduced expression of cyclin A. Conversely, FBI-1 repressed the transcription of p21CIP1 by inhibiting transcriptional activation of p53 and Sp1 in HeLa cells (39). Thus, FBI-1/OCZF/LRF affects the regulation of the cell cycle. In our study, OCZF decreased p21CIP1 expression in precursor cells, but increased expression of p21CIP1 and p27KIP1 in osteoclasts, suggesting that FBI-1/OCZF/LRF has different roles in proliferating osteoclast precursors and mature osteoclasts. Because RANKL-induced Cdk inhibitors are required for cell cycle withdrawal and differentiation in osteoclasts (40), the stimulatory effect of FBI-1/OCZF/LRF on Cdk inhibitor transcription may promote osteoclastogenesis.

Osteoclast formation is induced by RANKL and several inflammatory cytokines, and mature osteoclasts rapidly undergo apoptotic cell death in the absence of trophic factors such as M-CSF or RANKL. Enhanced osteoclast survival in OCZF-Tg mice suggests that OCZF prevents apoptosis of osteoclasts. In the absence of RelA, which has an antiapoptotic effect in osteoclasts, bone destruction in arthritis was markedly reduced in mice (41). Therefore, antiapoptotic factors may increase the number of osteoclasts and the process of bone resorption.

Interestingly, Maeda et al reported that LRF regulates apoptosis of erythroblasts, which is mediated by a proapoptotic protein, the BH3-only protein Bim (13). Expression of Bim is markedly up-regulated in the course of osteoclast apoptosis (42, 43). Therefore, it is important to examine whether Bim is involved in the survival-promoting activity of FBI-1/OCZF/LRF in osteoclasts.

POK family proteins are transcriptional regulators that recruit transcriptional corepressors such as histone deacetylases (HDACs) into complexes at the target gene promoter region (14). The amino-terminal POZ domain of FBI-1 binds HDAC-1 to form a repressor complex during adipogenesis, thereby repressing the transcription of cyclin A and E2F-4 (37). Inhibition of HDAC activity may affect osteoclast formation and bone resorption, a process in which FBI-1/OCZF/LRF may be implicated. Consistent with this hypothesis, we found that HDAC inhibitors, such as sodium butyrate or trichostatin A, selectively inhibited osteoclastogenesis in vitro (44). We further found that another HDAC inhibitor, FR901228, selectively inhibited osteoclastogenesis and bone destruction in rats with AIA (44, 45).

We have thus shown that the transcription factor FBI-1/OCZF/LRF is an important positive regulator of RANKL-induced osteoclastogenesis in vivo. It is localized specifically in the nuclei of osteoclasts and should prove useful as an immunohistochemical marker for osteoclasts in bone tissue and bone destruction in inflammatory disease. Thus, by enhancing osteoclastogenesis and survival of osteoclasts, FBI-1/OCZF/LRF might play important roles in bone turnover and bone destruction in vivo, including in the bone loss and remodeling that occurs in chronic inflammatory arthropathies such as RA.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. A. Kukita had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. A. Kukita, T. Kukita, Pessler, Shobuike.

Acqusition of data. A. Kukita, T. Kukita, Nagata, Teramachi, Li, Gay, Pessler, Shobuike.

Analysis and interpretation of data. A. Kukita, T. Kukita, Yoshida, Miyamoto, Gay, Pessler, Shobuike.

Acknowledgements

We thank Dr. T. Maeda for donating the anti–FBI-1/LRF IgG, Dr. M. Ouchida for providing the constructs of the pΔTK-RL vector, Drs. H. Takayanagi and M. Asagiri for helpful suggestions and discussion, and Dr. K. Matsuo for a critical reading of the manuscript and helpful suggestions. The human tissue specimens were obtained at the University of Zürich as part of an ongoing study of human synovitis that has been approved by the local Ethics Committee.

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