Blimp1-mediated repression of negative regulators is required for differentiation

Keizo Nishikawaa,b, Tomoki Nakashimaa,b,c, Mikihito Hayashia,b, Takanobu Fukunagaa,b, Shigeaki Katod,e, Tatsuhiko Kodamaf, Satoru Takahashig, Kathryn Calameh, and Hiroshi Takayanagia,b,c,1

aDepartment of Cell Signaling, Tokyo Medical and Dental University, Tokyo 113-8549, Japan; bGlobal Center of Excellence Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo 113-8549, Japan; cTakayanagi Osteonetwork Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Tokyo 113-8549, Japan; dInstitute of Molecular and Cellular Biosciences, Graduate School of Medicine, University of Tokyo, Tokyo 113-0032, Japan; eKato Nuclear Complex, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan; fResearch Center for Advanced Science and Technology, Department of Molecular Biology and Medicine, University of Tokyo, Tokyo 153-8904, Japan; gInstitute of Basic Medical Sciences and Laboratory Animal Resource Center, University of Tsukuba, Tsukuba 305-8575, Japan; and hDepartment of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY 10032

Edited by Anjana Rao, Harvard Medical School, Boston, MA, and approved December 23, 2009 (received for review November 7, 2009) Regulation of irreversible cell lineage commitment depends on a own promoter in cooperation with c-Fos (12). This autoampli- delicate balance between positive and negative regulators, which fication mechanism enables a robust induction of Nfatc1 specific comprise a sophisticated network of factors. Recep- to (13). tor activator of NF-κB ligand (RANKL) stimulates the differentia- During osteoclastogenesis, NFATc1 is constantly activated by tion of bone-resorbing osteoclasts through the induction of calcium signaling, which is dependent on Ig-like receptors such nuclear factor of activated T cells c1 (NFATc1), the essential tran- as osteoclast-associated (OSCAR) and triggering scription factor for osteoclastogenesis. Osteoclast-specific robust receptor expressed in myeloid cells-2 (TREM2) (1, 2). These induction of NFATc1 is achieved through an autoamplification receptors are associated with the adaptor molecules, DNAX- mechanism, in which NFATc1 is constantly activated by calcium activating 12 (DAP12) and Fc receptor common γ sub- signaling while the negative regulators of NFATc1 are suppressed. unit (FcRγ), both of which contain the immunoreceptor tyrosine- However, it has been unclear how such negative regulators are based activation motif (ITAM) in their cytoplasmic domain (14, repressed during osteoclastogenesis. Here we show that B lym- 15). Spleen tyrosine kinase (Syk) is recruited to the tyrosine- phocyte-induced maturation protein-1 (Blimp1; encoded by phosphorylated ITAM and makes a complex with the Tec family Prdm1), which is induced by RANKL through NFATc1 during osteo- kinases activated by RANK, which efficiently induces phospho- clastogenesis, functions as a transcriptional of anti- lipase Cγ (PLCγ) phosphorylation leading to the activation of Irf8 Mafb osteoclastogenic such as and . Overexpression of calcium signal through inositol triphosphate (16). Prdm1 Blimp1 leads to an increase in osteoclast formation, and - NFATc1 functions as the key regulator of osteoclast differ- fi de cient osteoclast precursor cells do not undergo osteoclast dif- entiation (13, 17, 18) by inducing fusogenic genes such as Tm7sf4 fi ferentiation ef ciently. The importance of Blimp1 in bone homeo- [encoding -specific transmembrane protein (DC-

stasis is underscored by the observation that mice with an MEDICAL SCIENCES fi fi Prdm1 STAMP)] (19, 20) and Atp6v0d2 (20, 21) in addition to a number osteoclast-speci cde ciency in the exhibit a high of genes (such as Acp5, Calcr, and Itgb3) expressed in mature bone mass phenotype caused by a decreased number of osteo- polarized osteoclasts that are involved in the regulation of bone- clasts. Thus, NFATc1 choreographs the determination of cell fate resorbing activity (1). Recent reports indicate that NFATc1 in the osteoclast lineage by inducing the repression of negative activity is negatively regulated by other transcription factors such regulators as well as through its effect on positive regulators. as IFN regulatory factor-8 (IRF-8) and v- musculoaponeur- otic fibrosarcoma oncogene family, protein B (MafB) during B lymphocyte induced maturation protein 1 | nuclear factor of activated T osteoclastogenesis (22, 23). The expression of such negative cells c1 | osteoclastogenesis | transcription | bone regulators was observed to be repressed through osteoclasto- genesis. This repression is consistent with the notion that high steoclasts are multinucleated cells of / NFATc1 activity is a prerequisite for efficient osteoclastogenesis Olineage that degrade bone matrix. The maintenance of bone (12), but the mechanism by which the expression of these anti- is dependent on the balance between bone-resorb- osteoclastogenic regulators is repressed during RANKL-induced ing osteoclasts and bone-forming osteoblasts (1, 2). Excessive osteoclastogenesis has remained obscure. bone resorption by osteoclasts is often associated with diseases To identify the negative transcriptional regulators in osteoclast accompanied by pathological bone loss, including osteoporosis differentiation, we performed a genome-wide screening of – and rheumatoid arthritis (3 6); thus research into the mecha- RANKL-inducible transcription factors and found that B lym- nisms of osteoclast differentiation is both biologically and phocyte-induced maturation protein-1 (Blimp1) (encoded by clinically important. Prdm1) is crucial for the repression of anti-osteoclastogenic κ Receptor activator of NF- B ligand (RANKL) is a key cyto- genes such as Irf8 and Mafb. Blimp1 is a transcriptional repressor kine that stimulates the osteoclast precursor cells to fuse and that plays crucial roles in the differentiation and/or function of undergo differentiation into osteoclasts (7, 8). Macrophage col- ony-stimulating factor (M-CSF) also is an essential cytokine for

osteoclastogenesis, which is important for the survival of the Author contributions: K.N., T.N. and H.T. designed research; K.N., M.H., T.F., S.K., T.K., S.T. osteoclast lineage and the expression of RANK, the receptor for and C.K. performed research; K.N. analyzed data; and K.N., T.N. and H.T. wrote the paper. RANKL (9). RANKL binding to RANK results in the recruit- The authors declare no conflict of interest. ment of the TNF receptor-associated factor 6 (TRAF6), leading This article is a PNAS Direct Submission. κ to the activation of NF- B and JNK (10). RANK also stimulates Data deposition: GeneChip data are available for download from the Genome Network the induction of Fos and thus activates the activator protein-1 Platform (http://genomenetwork.nig.ac.jp/). (AP-1) complex containing c-Fos (11). NF-κB induces the initial 1To whom correspondence should be addressed. E-mail: [email protected]. induction of nuclear factor of activated T cells c1 (Nfatc1), the This article contains supporting information online at www.pnas.org/cgi/content/full/ expression of which is autoamplified by NFATc1 binding to its 0912779107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0912779107 PNAS | February 16, 2010 | vol. 107 | no. 7 | 3117–3122 Downloaded by guest on October 1, 2021 various kinds of cells such as and lymphocytes (24). A In particular, Blimp1 is necessary for B cells to undergo terminal differentiation into plasma cells through direct repression of transcription factors such as Pax5, Bcl6, and , which are specifically important in the lineage commitment and pro- liferation of B cells (24, 25). Here we show that Blimp1 is induced by RANKL through NFATc1 and functions as a global repressor of negative regu- lators of NFATc1 during osteoclastogenesis. We also provide genetic evidence that Blimp1 is important for bone homeostasis by its promotion of osteoclastogenesis. These results reveal a previously undescribed aspect of the NFATc1-mediated tran- scriptional program of osteoclastogenesis, the repression of negative regulators by an NFATc1 target gene. Results and Discussion Genome-Wide Screening of Transcription Factors Involved in Osteoclastogenesis. To explore the transcription factors involved in osteoclast differentiation, we performed a genome-wide screening of mRNAs expressed in bone marrow-derived mono- cyte/macrophage precursor cells (BMMs) stimulated by B RANKL. We clustered the data of 1,675 transcription factors (26) using k-means algorithm into 22 groups (27). Four clusters of genes exhibited a common expression pattern characterized by a steady increase (by more than 2-fold at each time point after RANKL treatment) (Fig. 1A, Upper). Clusters I, II, III, and IV contained 72, 54, 48, and 7 transcription factors, respectively (Fig. 1A, Upper). From these four clusters, we finally selected 13 C D genes that are highly expressed in mature osteoclasts (an average difference >500 at 3 days) (Fig. 1A, Lower). Among the 13 genes selected were Nfatc1, Ppargc1b, and Fosl2, which are known to function as transcriptional activators in osteoclast differentiation (13, 28, 29). Blimp1 also has been classified with this group of transcription factors but is known to be a transcriptional repressor required for function (24). We confirmed that both the protein and mRNA expression of Prdm1 increased markedly during osteoclastogenesis (Fig. 1 B E and C). Prdm1 was dramatically induced in BMMs stimulated by RANKL in the presence of M-CSF (Fig. 1C). It has been reported that Prdm1 expression increases with the macrophage differentiation induced by phorbol-12-myristate 13-acetate (30), but M-CSF treatment alone did not induce the Prdm1 expression in this time course. To test whether Prdm1 induction by RANKL is dependent on NFATc1, we investigated the Prdm1 expression in the presence of a calcineurin inhibitor, cyclosporin A. As Fig. 1. Increased expression of Prdm1 during osteoclastogenesis. (A) Gen- expected, Prdm1 induction was greatly suppressed by the treat- eChip analysis of expression during osteoclast differ- ment with cyclosporin A, suggesting that Prdm1 expression is entiation. Among the 22 clusters, clusters I to IV were characterized by a regulated by NFATc1 (Fig. 1D). We performed a computational steady increase. The increased mainly in the early (I), search on the sequence from 5 kb upstream to 5 kb downstream intermediate (II), or late (III) phase, or continuously (IV). The log-transformed of the transcription start site of the Prdm1 gene and found an value of the mRNA expression of each gene is depicted by a black line, and the average of the black lines is shown in red. (B) Immunoblot analysis to NFATc1-binding site in a region conserved in the human and detect the expression of Blimp1 protein in BMMs stimulated with RANKL in mouse sequences (Fig. 1E). Indeed, ChIP analysis showed that the presence of M-CSF. (C) mRNA expression of Prdm1 in BMMs cultured NFATc1 binds directly to the site in BMMs stimulated with with or without RANKL. (D) Effect of cyclosporin A (CsA; 0.8 μM) on the RANKL for 2 days but not in unstimulated BMMs (Fig. 1E). expression of Prdm1 in BMMs stimulated with RANKL for 2 days. **, P < 0.01. These results suggest that Prdm1 is a direct transcriptional target (E) Recruitment of NFATc1 to the Prdm1 promoter region. Schematic view of of NFATc1 during osteoclastogenesis. the NFATc1-binding site in the regulatory region of Prdm1 (expanded, Lower Left). DNA sequences conserved between the mouse and human sequences Repression Activity of Blimp1 Is Important for Osteoclast were identified using VISTA Genome Browser (http://genome.lbl.gov/vista/ Differentiation. Although it has been reported previously that index.shtml). Arrowheads indicate the primer set used for ChIP. The sites Blimp1 functions as a transcriptional repressor in other cell types colored red represent the NFATc1-binding site. ChIP assay and representative results (Lower Right). (24, 25), transcription factors can function as either a positive or a negative transcriptional regulator in a context-dependent manner (31). To investigate whether Blimp1 acts as a transcriptional activator or repressor during osteoclast differentiation, we con- and RD-Blimp1, respectively) (Fig. 2A) and overexpressed these structed Blimp1 variants that constitutively function as a tran- variants in BMMs using the retrovirus vector (Fig. 2B) to test their scriptional activator or repressor (32). We fused either the effects on osteoclast formation. The introduction of RD-Blimp1 transactivation domain of herpes simplex virus VP16 or the into BMMs by retroviral transfer led to increased osteoclast dif- repressor domain of Drosophila to Blimp1 (AD-Blimp1 ferentiation, whereas the overexpression of AD-Blimp1 inhibited

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B Fig. 2. Blimp1 functions as a transcriptional repressor in the promotion of osteoclast differentiation. (A) Schematic view of WT Blimp1, RD-Blimp1, and AD-Blimp1. (B) mRNA expression of RD-Blimp1 and AD-Blimp1 after infec- tion with pMX-RD-Blimp1-IRES-EGFP (pMX-RD-Blimp1) and pMX-AD-Blimp1- IRES-EGFP (pMX-AD-Blimp1), respectively. (C) Effects of retroviral expression of RD-Blimp1 or AD-Blimp1 on the osteoclastogenesis from BMMs stimu- lated with RANKL. *, P < 0.05; **, P < 0.01. (D) Osteoclast formation in BMMs transduced with pMX-RD-Blimp1 or pMX-AD-Blimp1.

osteoclast formation (Fig. 2 C and D). These results suggest that Blimp1 functions mainly as a transcriptional repressor in the promotion of osteoclast differentiation. C MEDICAL SCIENCES

High Bone Mass Phenotype Induced by Osteoclast-SpecificDeficiency of Prdm1. Because Prdm1-deficient mice are embryonically lethal (25), to investigate the role of Blimp1 in osteoclasts in vivo,we fl – crossed Prdm1 ox/ mice (25) with CtskCre/+ mice (33) to disrupt Δ – the Prdm1 gene specifically in the osteoclast lineage (Prdm1 OC/ ). Δ − Microcomputed tomography clearly indicated that the bone Fig. 3. Prdm1 OC/ mice exhibit a high bone mass phenotype. (A) Micro- ΔOC/– fl fl volume was greatly enhanced in Prdm1 mice at the age of 12 computed tomography (μCT) analysis of the femurs of Prdm1 ox/ ox and Δ – and 32 weeks (Fig. 3A). The osteoclast number was significantly Prdm1 OC/ mice (Upper photograph: longitudinal view; Lower photograph: reduced in the metaphyseal region, and the bone marrow was axial view of the metaphyseal region). The parameters are based on the μCT Δ – abnormally filled with trabecular bone in Prdm1 OC/ mice (Fig. analysis of the metaphyseal region. *, P < 0.05; **, P < 0.01; N.S., not sig- nificant. (B) Histological analysis of the proximal tibia of Prdm1flox/flox and 3 B and C). Bone morphometric analysis indicated an increase in Δ − Prdm1 OC/ mice (Left, toluidine blue staining, purple marks cartilage; Cen- bone volume associated with a reduced osteoclast number and a ter, von Kossa staining, black indicates mineralized bone; Right, TRAP decrease in the indicators of osteoclastic bone resorption (Fig. staining, red marks osteoclasts). (C) Parameters for osteoclastic bone 3C). Osteoblastic parameters were slightly decreased in resorption and osteoblastic bone formation in the bone morphometric ΔOC/– fl fl Δ – Prdm1 mice (Fig. 3C), possibly because of an osteoclast– analysis of Prdm1 ox/ ox and Prdm1 OC/ mice at the age of 32 weeks. *, P < osteoblast coupling mechanism. These results collectively suggest 0.05; **, P < 0.01; N.S., not significant. Δ – that the increase in bone mass in Prdm1 OC/ mice was caused by impaired osteoclastic bone resorption owing to a defect in osteoclast differentiation. Having normal tooth eruption, the genomic PCR analysis indicates that the Prdm1 gene was excised Δ – fi Prdm1 OC/ mice exhibit an incomplete osteopetrotic phenotype, ef ciently 2 days after RANKL treatment (Fig. 4B). Consistently, the protein and mRNA expression of Prdm1 was diminished in but the results nevertheless suggest that Blimp1 plays a critically ΔOC/– important role in osteoclast differentiation. Prdm1 cells 2 days after RANKL treatment (Fig. 4 C and D). These results indicate that the Prdm1 gene was disrupted in the Impaired Osteoclastogenesis in Prdm1-Deficient Cells. In vitro middle stage of osteoclast differentiation (24–48 h after RANKL osteoclast differentiation was evaluated by counting the multi- stimulation in the 72-h period of differentiation). There was no nucleated cells (MNCs) positive for the osteoclast marker tar- significant difference in the number of CD11b+ cells in the BMMs Δ – trate-resistant acid phosphatase (TRAP) after stimulation of (Fig. 4E), suggesting that Prdm1 OC/ cells contain a normal BMMs with RANKL in the presence of M-CSF. The number of number of osteoclast precursor cells. RANKL-stimulated induc- Δ – TRAP-positive MNCs was markedly reduced in the Prdm1 OC/ tion of the osteoclast-specific genes Nfatc1, Acp5, and Ctsk was fl fl Δ – cells compared with the Prdm1 ox/ ox cells (Fig. 4A). Quantitative severely reduced in Prdm1 OC/ cells (Fig. 4D).

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Δ – Fig. 4. Impaired osteoclastogenesis in Prdm1 OC/ cells. (A) Osteoclast dif- fl fl Δ – ferentiation in Prdm1 ox/ ox and Prdm1 OC/ BMMs stimulated with RANKL. **, P < 0.01. (B) Quantitative genomic PCR analysis to detect the recombi- nation efficiency in Prdm1flox/flox and Prdm1ΔOC/– BMMs stimulated by fl fl Δ – RANKL. (C) Protein expression of Blimp1 in Prdm1 ox/ ox and Prdm1 OC/ BMMs stimulated with RANKL. (D) Real-time RT-PCR analysis of mRNAs for fl fl Δ – Fig. 5. Blimp1 regulates osteoclast differentiation through the down- Nfatc1, Acp5, Ctsk, and Prdm1 in Prdm1 ox/ ox and Prdm1 OC/ BMMs regulation of multiple negative regulators. (A) Comprehensive search for stimulated with RANKL. **, P < 0.01. (E) Expression of CD11b in the fl fl Δ – the Blimp1-regulated genes during osteoclastogenesis. The expression of Prdm1 ox/ ox and Prdm1 OC/ BMMs (flow cytometry). (F) Effect of retroviral Δ – 365 genes decreased more than 2-fold every 24 h during osteoclast dif- expression of HA-tagged Blimp1 on osteoclastogenesis on Prdm1 OC/ BMMs ferentiation (Left). Of these genes, we identified the expression of 116 stimulated with RANKL. **,P < 0.01. genes to be sufficiently recovered (>120%) in Prdm1-deficient cells (Cen- ter). Prdm1-deficient cells were generated by retroviral introduction of Cre fl fl recombinase into Prdm1 ox/ ox BMMs. A computational search for the To evaluate the effect of Blimp1 overexpression, we con- Blimp1-binding site in the region conserved between the human and structed a retroviral vector carrying the HA-tagged Blimp1 gene. mouse sequences within 500 kb upstream of the transcriptional start site of Δ – We transduced the Prdm1 OC/ cells with the viral vector and 116 genes using the ECR Browser (http://ecrbrowser.dcode.org/) showed evaluated the TRAP-positive MNC formation. Impaired osteo- that 60 genes, including eight transcription factors, have a putative Blimp1- Δ – clast formation in Prdm1 OC/ cells was significantly restored by binding site (Right). The number in parentheses indicates the number of Blimp1-binding sites. (B) Recruitment of Blimp1 to the Irf8 and Mafb reg- the overexpression of Blimp1 (Fig. 4F), demonstrating that ulatory regions. Schematic view of the Blimp1-binding site in the regulatory Blimp1 has an ability to promote osteoclastogenesis. region of Irf8 and Mafb (Upper). BMMs cultured with RANKL and M-CSF for 2 days were used. Arrowheads indicate the primer sets used for ChIP. Sites Blimp1 Represses Expression of the Irf8 and Mafb Genes in colored red represent Blimp1-binding sites. ChIP assay and representative Osteoclasts. What are the target(s) of Blimp1-mediated repres- results (Lower). (C) Real-time RT-PCR analysis of Irf8 and Mafb mRNAs in flox/flox ΔOC/– sion in the promotion of osteoclastogenesis? To identify the Prdm1 and Prdm1 BMMs stimulated with RANKL for 2 days. *, P < < putative Blimp1-regulated genes, we performed a genome-wide 0.05; **, P 0.01. (D) Real-time RT-PCR analysis of Irf8 and Mafb mRNAs in HA-tagged Blimp1-expressed BMMs stimulated with RANKL for 2 days. **, P screening of mRNAs during osteoclastogenesis using WT and < Δ Δ 0.01. (E) A model of Blimp1-mediated regulation of osteoclast differ- Prdm1 OC/ OC BMMs (Fig. 5A, Left and Center). We first

3120 | www.pnas.org/cgi/doi/10.1073/pnas.0912779107 Nishikawa et al. Downloaded by guest on October 1, 2021 selected genes the expression of which was decreased more than Accumulating evidence indicates that immune and bone cells 2-fold every 24 h during osteoclast differentiation in WT cells share important regulatory molecules, including receptors, sig- (Fig. 5A, Left). We further selected genes in which the reduced naling molecules, enzymes, and transcription factors. These expression was clearly normalized by the Prdm1 deficiency (Fig. findings have shed light on the new interdisciplinary research 5A, Center). We computationally searched the 5′-flanking region field of osteoimmunology (1, 2). In particular, an unexpectedly of these genes for the Blimp1-binding site to identify 60 genes high number of factors that regulate osteoclast differentiation having a putative Blimp1-binding site (Fig. 5A, Right). The eight were identified originally in immune cells (1, 2). Blimp1 also has transcription factors were included in these genes (Fig. 5A, been well characterized as a repressor of genes involved in early Right). In this study, we focused on Irf8 and Mafb because these B-cell differentiation that enable B cells to undergo terminal genes are reported to be negative regulators of osteoclast dif- differentiation into plasma cells (24). The function of Blimp1 in ferentiation (22, 23). We identified nine Blimp1-binding sites in osteoclasts is analogous to its role in plasma cells, in that Blimp1 the 5′-flanking sequence of Irf8 and three Blimp1-binding sites in represses the genes involved in macrophage development such as the 5′-flanking sequence of Mafb. A cluster of Blimp1-binding Irf8 and Mafb (35, 36), thus converting monocyte/macrophage sites is located ≈5.5 kb upstream from the transcription start site precursor cells into terminally differentiated osteoclasts. of the Irf8 gene. Indeed, ChIP analysis showed that Blimp1 Because Blimp1 acts as a global repressor of multiple anti- bound to the sequence containing the cluster in the 5′-flanking osteoclastogenic genes, it is possible that inhibitors of Blimp1 sequence of Irf8 (Fig. 5B). Blimp1 also bound to one of the three function may serve as antiresorptive agents by inducing various Blimp1-binding sites located ≈16 kb upstream from the tran- factors affecting osteoclastogenesis. In depth understanding of scription start site of the Mafb gene (Fig. 5B). These results the gene regulatory programs mediated by Blimp1 should help suggest that Blimp1 represses the expression of Irf8 and Mafb by provide a molecular basis for future therapeutic strategies in directly binding to their regulatory regions. Consistent with this bone disease. notion, the expression of Irf8 and Mafb was increased in Δ – Prdm1 OC/ cells (Fig. 5C), and the forced expression of Blimp1 Materials and Methods decreased the expression of Irf8 and Mafb significantly (Fig. 5D). Mice and Analysis of Bone Phenotype. Prdm1flox/– and CtskCre/+ mice were These results suggest that the Irf8 and Mafb genes are among the generated previously and have been described elsewhere (25, 33). Both direct repressive targets of Blimp1 during osteoclastogenesis. strains were backcrossed with C57/BL6 mice for more than six generations. To investigate whether Blimp1 promotes osteoclastogenesis by All mice were born and maintained under specific-pathogen–free con- the down-regulation of Irf8 and Mafb, we generated retroviral ditions. All animal experiments were performed with the approval of the Animal Study Committee of Tokyo Medical and Dental University and con- vectors carrying Irf8 or Mafb shRNA. However, the introduction ΔOC/– formed to relevant guidelines and laws. CT scanning was performed using a of Irf8 and/or Mafb shRNAs into Prdm1 BMMs had only a ScanXmate-A100S Scanner (Comscantechno). Three-dimensional micro- minimal rescue effect (Fig. S1), suggesting that Blimp1 exerts its structural image data were reconstructed and structural indices were cal- function through the regulation of multiple genes, including Irf8 culated using TRI/3D-BON software (RATOC Systems). Bone morphometric and Mafb. Pax5 expression is known to be repressed by Blimp1 in analysis was performed as described (14, 16). − − plasma cells (24), and Pax5 / mice exhibit an osteopenic phe- notype because of an increased number of osteoclasts (34), Cell Cultures. The method for in vitro osteoclast differentiation was described previously (13, 14, 16). In brief, bone marrow cells cultured with 10 ng mL−1

suggesting that Pax5 is a candidate target of Blimp1 in osteo- MEDICAL SCIENCES M-CSF (R&D Systems) for 2 days were used as BMMs, which were further clasts. However, the contribution of Pax5 to the Blimp1-medi- − cultured with 50 ng mL 1 RANKL (Peprotech) in the presence of M-CSF (10 ng ated regulation of osteoclastogenesis may be minimal, because −1 the expression of Pax5 is negligible during osteoclastogenesis mL ) for 3 days. based on the GeneChip data. Bcl6 also is repressed by Blimp1, Flow Cytometric Analysis. Two days after stimulation with RANKL, cells were and Blimp1 is negatively regulated by BCL6 in plasma cells (24). stained with the PE-conjugated anti-CD11b (BD Biosciences) or Although the shRNA-mediated knockdown of Bcl6 did not control rat IgG. Stained cells were analyzed with a flow cytometer (FACS- ΔOC/– normalize the decreased osteoclastogenesis in Prdm1 cells Canto II; BD Biosciences). (Fig. S1), we cannot rule out the possibility that Bcl6 may also contribute to the Blimp1-mediated regulation of osteoclasto- Quantitative PCR Analysis. Total RNA and cDNA were prepared by ISOGEN genesis in cooperation with Irf8 and Mafb. (WakoChemicals)andSuperScriptIIIreversetranscriptase(Invitrogen)according Cell lineage commitment is strictly regulated to maintain the to the manufacturer’s instructions. Genomic DNA was obtained by phenol/ homeostasis of the biological systems of the multicellular chloroform extraction and ethanol precipitation. Quantitative PCR analysis was organisms. To this end, cell differentiation signals usually stim- performed with a LightCycler (Roche) using SYBR Green (Toyobo). The primer ′ ulate the negative-feedback regulatory pathways to correct any sequences used in real-time RT-PCR analysis were Actb,5-CTTCTACAATG- AGCTGCGTG-3′ and 5′-TCATGAGGTAGTCTGTCAGG-3′; Acp5, 5′-GGGAAAT- excess. During osteoclastogenesis, however, NFATc1 activity and GGCCAATGCCAAAGAGA-3′ and 5′-TCGCACAGAGGGATCCATGAAGTT-3′; Ctsk, expression are maintained at an extremely high level compared 5′-AGGCAGCTAAATGCAGAGGGTACA-3′ and 5′-ATGCCGCAGGCGTTGTTCTT- with other cell types (12). Therefore, the expression of molecules ATTC-3′; Nfatc1, 5′-GGTAACTCTGTCTTTCTAACCTTAAGCTC-3′ and 5′-GTGAT- that inhibit NFATc1 activity needs to be repressed. Blimp1, GACCCCAGCATGCACCAGTCACAG-3′; Prdm1, 5′-TGCTTATCCCAGCACCCC-3′ which is induced by the RANKL–NFATc1 axis, represents the and 5′-CTTCAGGTTGGAGAGCTGACC-3′; Mafb, 5′-AACGGTAGTGTGGAGGAC- repressor of anti-osteoclastogenic genes (Fig. 5E). It is likely that 3′ and 5′-TCACAGAAAGAACTCAGGA-3′; Irf8, 5′-AAGGTCACCGTGGTCCTTAG- NFATc1 functions as the crucial regulator of osteoclastogenesis 3′ and 5′-GGAAAGCCTTACCTGCTGAC-3′; engrailed, 5′-TCGAATTCAAGG- both by inducing osteoclastogenic genes and by acting as a TATGGCCCTGGAG-3′ and 5′-GGGAATTCGCTCGAGAGGGATCCCAGAGCA-3′; ′ ′ ′ repressor of negative regulatory genes. VP16, 5 -GGGAATTCGGATCCCATCGATATGACCGGA-3 and 5 -GGGAATTC- TTCTAGAGGCTCGAGCCCACCG-3′. The primer sequences used in quantitative genomic PCR analysis were Prdm1 third exon, 5′-gctgcccccaagtctagc-3′ and 5′-cttcaaactcggcctctgtcc-3′; entiation. During osteoclastogenesis, the critical transcription factor Prdm1 sixth intron/seventh exon, 5′-aagaggcttgcatctgcatt-3′ and 5′-tgtgg- NFATc1 is induced by RANKL and ITAM signals. NFATc1 is known to regu- cttctctcctgtgtg-3′. late various osteoclastogenic genes. Anti-osteoclastogenic transcription factors such as IRF-8 and MafB inhibit osteoclast differentiation mainly GeneChip Analysis. GeneChip analysis was performed as described (13, 14, 16). through the suppression of NFATc1 activity. Blimp1, which also is induced Briefly, the total RNAs were used for cDNA synthesis by reverse transcription by the RANKL-NFATc1 axis, represents the repressor of anti-osteoclasto- followed by synthesis of biotinylated cRNA through in vitro transcription. genic genes. After cRNA fragmentation, hybridization with mouse genome 430 2.0 array

Nishikawa et al. PNAS | February 16, 2010 | vol. 107 | no. 7 | 3121 Downloaded by guest on October 1, 2021 (Affymetrix) was performed. The main part of the dataset has been depos- Knockdown Analysis. We used the RNAi system (BD Biosciences) and con- ited and can be obtained from the Genome Network Platform (http:// structed the Irf8 and Mafb RNAi-Ready pSIREN vector according to the man- genomenetwork.nig.ac.jp/). K-means clustering analysis of gene expression ufacturer's protocol. Briefly, annealed oligonucleotides (Irf8 RNAi sense: on their Euclidean distance was performed with the Cluster 3.0 (27). gatcCCGCTGAAATGGAGTTAGTTTCTCGAGAAACTAACTCCATTTCAGCGGTTT- TTG; Irf8 RNAi antisense: aattCAAAAACCGCTGAAATGGAGTTAGTTTCTCGA- ChIP Assay. ChIP assay was performed by the ChIP Assay Kit (Upstate) GAAACTAACTCCATTTCAGCGG; Mafb RNAi sense: gatcGCTGAGTCTTTG according to the manufacturer's instructions with minor modifications. In TTTGGGTTTCTCGAGAAACCCAAACAAAGACTCAGCTTTTTG; Mafb RNAi anti- brief, BMMs stimulated with RANKL for 2 days were fixed with form- sense: aattCAAAAAGCTGAGTCTTTGTTTGGGTTTCTCGAGAAACCCAAACAAA aldehyde. After quenching with glycine, cells were lysed and sonicated. GACTCAGC; Bcl6 RNAi sense: gatcGCTGTCAAAGAGAAGGCTTTACTCGAGT Sonicated chromatin was incubated with either rabbit anti-Blimp1 poly- AAAGCCTTCTCTTTGACAGCTTTTTG; Bcl6 RNAi antisense: aattCAAAAAGCTGT clonal antibody serum (37) or anti-NFATc1 antibody (Santa Cruz). We used CAAAGAGAAGGCTTTACTCGAGTAAAGCCTTCTCTTTGACAGC) were ligated rabbit serum or rabbit IgG (Santa Cruz) as a control. Immunoprecipitation into a pSIREN vector. After 12-h inoculation with retroviruses carrying the pSIREN vector, BMMs were stimulated with RANKL followed by selection with was performed using protein A Dynabeads (Invitrogen). After extensive − 2.5 μgmL 1 puromycin (Invitrogen). washing, protein-DNA crosslinks were reversed, and the precipitated DNA was treated with proteinase K before phenol chloroform extraction and ethanol precipitation. The primer sequences were Prdm1, 5′- Immunoblot Analysis. Immunoblot analysis was performed using against Blimp1 (NOVUS), HA (Abgent), and Laminb1 (Santa Cruz) as previously AAACGTGTGGGTACGACCTT-3′ and 5′-AGCGCTGGTTTCTACTGAGG-3′; Irf8, described (40). Anti-Laminb1 antibody was used as an internal control. 5′-TCCCTCCCTCCTTCTCCTTA-3′ and 5′-AAGCCCTGAGTGCACAGACT-3′; Mafb site 1, 5′-CAGAGGCAAAGGAACAGCTC-3′ and 5′-GCACCCTGGACTGTAAC- CAT-3′; Mafb site 2, 5′-CCTTGCCTTGTGTCCTGAAG-3′ and 5′-GAGGGGG- Statistical Analysis. Statistical analysis was performed using the unpaired two- tailed Student's t test. All data are expressed as the mean ± SEM. TATGAAGGAGAGG-3′; Mafb site 3, 5′-GCGTCAGCTTCCCAGTAGAA-3′ and 5′- CGACTGGGAGCGTAGTTAGC-3′. ACKNOWLEDGMENTS. We thank T. Ando, Y. Kunisawa, M. Shinohara, M. Oh-hora, A. Izumi, and A. Hirota for discussion and technical assistance. We Plasmid Construction and Retroviral Gene Transfer. A retroviral vector, pMX- also thank M. Kobayashi for the gift of pCS2-En and pCS2-VP16. This work HA-Blimp1, was constructed by inserting DNA fragments encoding HA and was supported in part by a grant to the Exploratory Research for Advanced Blimp1 into a retroviral expression vector, pMx-IRES-GFP (38). The retroviral Technology Takayanagi Osteonetwork Project from Japan Science and vectors pMX-RD-HA-Blimp1 and pMX-AD-HA-Blimp1 were constructed by Technology Agency; Grants-in-Aid for Creative Scientific Research and inserting DNA fragments encoding engrailed or VP16 (32) into pMX-HA- Young Scientist (A and Start-up) from the Japan Society for the Promotion Blimp1. A retroviral vector, pMX-Cre, has been described elsewhere (39). of Science (JSPS); a Grant-in-Aid for Challenging Exploratory Research from the JSPS; grants for the Genome Network Project and Global Center of Retroviral packaging was performed by transfecting the plasmids into Plat-E Excellence Program from the Ministry of Education, Culture, Sports, Science cells using FuGENE 6 (Roche) as described (38). Retroviral particles were used and Technology of Japan; and by grants from the Tokyo Biochemical for infection after concentration by centrifugation at 8,000 × g for 16 h. Research Foundation, the Life Science Foundation of Japan, the Yokoyama After 12-h inoculation, BMMs were stimulated with RANKL. Foundation for Clinical Pharmacology, and the Takeda Science Foundation.

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