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. 2010 Apr 12;207(4):751-62.
doi: 10.1084/jem.20091957. Epub 2010 Apr 5.

The Blimp1-Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis

Yoshiteru Miyauchi  1 , Ken Ninomiya, Hiroya Miyamoto, Akemi Sakamoto, Ryotaro Iwasaki, Hiroko Hoshi, Kana Miyamoto, Wu Hao, Shigeyuki Yoshida, Hideo Morioka, Kazuhiro Chiba, Shigeaki Kato, Takeshi Tokuhisa, Mitinori Saitou, Yoshiaki Toyama, Toshio Suda, Takeshi Miyamoto
Affiliations

The Blimp1-Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis

Yoshiteru Miyauchi et al. J Exp Med. .

Abstract

Controlling osteoclastogenesis is critical to maintain physiological bone homeostasis and prevent skeletal disorders. Although signaling activating nuclear factor of activated T cells 1 (NFATc1), a transcription factor essential for osteoclastogenesis, has been intensively investigated, factors antagonistic to NFATc1 in osteoclasts have not been characterized. Here, we describe a novel pathway that maintains bone homeostasis via two transcriptional repressors, B cell lymphoma 6 (Bcl6) and B lymphocyte-induced maturation protein-1 (Blimp1). We show that Bcl6 directly targets 'osteoclastic' molecules such as NFATc1, cathepsin K, and dendritic cell-specific transmembrane protein (DC-STAMP), all of which are targets of NFATc1. Bcl6-overexpression inhibited osteoclastogenesis in vitro, whereas Bcl6-deficient mice showed accelerated osteoclast differentiation and severe osteoporosis. We report that Bcl6 is a direct target of Blimp1 and that mice lacking Blimp1 in osteoclasts exhibit osteopetrosis caused by impaired osteoclastogenesis resulting from Bcl6 up-regulation. Indeed, mice doubly mutant in Blimp1 and Bcl6 in osteoclasts exhibited decreased bone mass with increased osteoclastogenesis relative to osteoclast-specific Blimp1-deficient mice. These results reveal a Blimp1-Bcl6-osteoclastic molecule axis, which critically regulates bone homeostasis by controlling osteoclastogenesis and may provide a molecular basis for novel therapeutic strategies.

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Figures

Figure 1.
Figure 1.
Bcl6 is suppressed during osteoclastogenesis and inhibits osteoclast formation. (A) Bcl6 expression was examined by comparative microarray analysis between osteoclast precursors (M-CSF) and osteoclasts (M-CSF + RANKL) cultured for 6 d. (B) BMMs were cultured with or without RANKL for 8 d and subjected to immunofluorescence staining (left) and immunoblot (right) for Bcl6. Nuclei were visualized by DAPI. Bar, 25 µm. (C) Recruitment of NFATc1 and Bcl6 to the NFATc1 P1 distal promoter was detected by ChIP assay. RAW264.7 cells were stimulated with or without RANKL for 48 h and subjected to ChIP analysis. (D) RAW264.7 cells transduced with Bcl6-overexpressing (Bcl6) or mock (control) retrovirus were cultured in the presence (RANKL) or absence (control) of RANKL for 5 d and stained with TRAP. Left, TRAP staining. (right) Numbers are means ± SD of multinuclear TRAP-positive cells in control or Bcl6-overexpressing RAW264.7 cells cultured with RANKL (**, P < 0.001; n = 6). Representative data of three independent experiments are shown (B–D).
Figure 2.
Figure 2.
Increased osteoclast formation resulting from Bcl6 deficiency. (A) Micro focus CT analysis of femurs of Bcl6+/− (left) and Bcl6−/− (right) mice. (B) Bone mineral density (BMD) of equal longitudinal division of femurs of Bcl6+/− (open circles) and Bcl6−/− (closed circles) mice. Data are mean BMD (mg/cm2) ± SD (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 3). (C) TRAP staining of tibial sections of Bcl6+/− (top) and Bcl6−/− (bottom) mice, and osteoclast surface as a percentage of bone surface (OcS/BS). Data are means ± SD (**, P < 0.001; n = 5). (D) Serum levels of C-terminal teropeptides of type I collagen (CTx) were analyzed in Bcl6+/− (white bar) and Bcl6−/− (shaded bar) mice. Data are means ± SD (**, P < 0.001; n = 3). (E) Osteoclast precursor cells from control (Bcl6+/−) or Bcl6−/− mice were cultured in the presence or absence of RANKL for 5 d and subjected to May-Grünwald Giemsa and TRAP staining, and the number of multinuclear TRAP-positive cells containing more than three nuclei was determined. Data are means ± SD of cells containing more than three nuclei (**, P < 0.001; n = 3). (F) Bone resorbing activity in Bcl6+/− (top) and Bcl6−/− (bottom) osteoclasts was analyzed by a pit formation assay. Representatives of at least three (E) and two (F) independent experiments are shown. Bars: (A) 1 mm; (C, E, and F) 100 µm.
Figure 3.
Figure 3.
Blimp1 is essential for osteoclastogenesis and regulates bone homeostasis. (A) Total RNA isolated from BMMs cultured with M-CSF and RANKL for indicated periods was subjected to RT-PCR analysis with primers specific for Blimp1 (top), Ctsk (middle), and β-actin (bottom). Representatives of at least three independent experiments are shown. (B–F) Bone phenotypes of osteoclast-specific Blimp1 KO (Blimp1 cKO) female mice at 8 wk old. (B) Longitudinal sections of tibias of control mice (Ctl; left) and Blimp1 cKO mice (right) were stained by toluidine blue. (C) Bone parameters are shown. Data are mean bone volume per total volume (BV/TV; %), trabecular number (Tb. N; /mm), osteoblast surface per bone surface (Ob.S/BS; %), or bone formation rate per bone surface (BFR/BS; mm3/cm2/y) ± SD of control (Ctl; white bar) and Blimp1 cKO (cKO; shaded bar) mice (*, P < 0.01; n = 5). (D) Microfocus CT analysis of femurs of control mice (Ctl; left) and Blimp1 cKO mice (right). (E) TRAP staining of tibial sections of control mice (Ctl; left) and Blimp1 cKO mice (right). Osteoclast parameters are shown as mean osteoclast number per bone perimeter (N.Oc/B.Pm) or osteoclast surface per bone surface (Oc.S/BS.) ± SD of control (Ctl; white bar) and Blimp1 cKO (cKO; shaded bar) mice (n = 5). (F) Serum CTx levels as a marker of bone resorption of control (Ctl; white bar) and Blimp1 cKO (cKO; shaded bar) mice (*, P < 0.01; n = 5). Bars: (B and E) 100 µm; (D) 1 mm.
Figure 4.
Figure 4.
Impaired osteoclastogenesis and Bcl6 expression resulting from Blimp1 deficiency. (A–C) BMMs from control (Ctl) or Blimp1 cKO (cKO) mice were cultured in the presence or absence of RANKL for 8 d. Cells were then subjected to May-Grünwald Giemsa and TRAP staining (A), the number of TRAP-positive cells containing more than three nuclei was scored (B, left), and the number of nuclei in each multinuclear osteoclast was determined (B, right). Bar, 100 µm. Numbers are means ± SD of multinuclear cells (**, P < 0.001; n = 3). (C) Total RNA was prepared from control (white bars) or Blimp1 cKO (shaded bars) cells treated with (+) or without (−) RANKL, and Blimp1 (left) or Bcl6 (right) expression relative to β-actin was analyzed by quantitative real-time PCR. Data represent means ± SD of Blimp1/β-actin or Bcl6/β-actin levels (**, P < 0.001; n = 4). (D) Whole-cell lysates from control or Blimp1 cKO cells cultured with M-CSF alone or M-CSF plus RANKL were analyzed by immunoblotting to detect Blimp1, Bcl6, and NFATc1. Actin was analyzed as an internal control. Representatives of at least four independent experiments are shown.
Figure 5.
Figure 5.
Bcl6 suppresses osteoclast differentiation. (A) Total RNA was prepared from control (white bars) or Blimp1 cKO (shaded bars) cells treated with (+) or without (−) RANKL, and the expression of the osteoclastic genes NFATc1, DC-STAMP, and Ctsk relative to β-actin was analyzed by a quantitative real-time PCR. Data are means ± SD of osteoclastic genes/β-actin. (**, P < 0.001; n = 4). (B) Recruitment of NFATc1 and Bcl6 to promoters of osteoclastic genes such as NFATc1, DC-STAMP, Ctsk, or a negative control gene NFATc2 was analyzed by a ChIP assay. Representatives of at least four (A) or two (B) independent experiments are shown.
Figure 6.
Figure 6.
Bcl6 is a target of Blimp1 during osteoclastogenesis. (A) Bone mineral density (BMD) of equal longitudinal division of femurs of control (filled circles), Blimp1 cKO (open circles), and Blimp1 cKO/Bcl6 KO (DKO; filled triangles) mice. Data represent mean BMD (n = 4). (B and C) Osteoclast progenitor cells from control, Blimp1 cKO or DKO mice were cultured in the presence of M-CSF alone or M-CSF plus RANKL for 8 d. Cells were then subjected to May-Grünwald Giemsa and TRAP staining (B), and the number of TRAP-positive cells containing more than three nuclei was scored (C). Results are representative of three independent experiments. Bar, 100 µm.
Figure 7.
Figure 7.
A schematic model of osteoclastogenesis regulated by the Blimp1-Bcl6-osteoclastic gene axis. RANKL–RANK interaction results in Blimp1 induction, leading to Bcl6 down-regulation and dissociation of Bcl6 from osteoclastic gene promoters, an event critical for osteoclastogenesis. NFATc1 activation is induced by various factors, such as ITAM, TRAF6, c-Fos, and Ca2+ signaling, which are also activated by RANKL.
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