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Identification of cell cycle-arrested quiescent osteoclast precursors in vivo.

Mizoguchi T, Muto A, Udagawa N, Arai A, Yamashita T, Hosoya A, Ninomiya T, Nakamura H, Yamamoto Y, Kinugawa S, Nakamura M, Nakamichi Y, Kobayashi Y, Nagasawa S, Oda K, Tanaka H, Tagaya M, Penninger JM, Ito M, Takahashi N - J. Cell Biol. (2009)

Bottom Line: Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D(3) analogue given to those mice.Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice.These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Oral Science, Matsumoto Dental University, Nagano 399-0781, Japan.

ABSTRACT
Osteoclasts are multinucleated cells that resorb bone. Although osteoclasts originate from the monocyte/macrophage lineage, osteoclast precursors are not well characterized in vivo. The relationship between proliferation and differentiation of osteoclast precursors is examined in this study using murine macrophage cultures treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB (RANK) ligand (RANKL). Cell cycle-arrested quiescent osteoclast precursors (QuOPs) were identified as the committed osteoclast precursors in vitro. In vivo experiments show that QuOPs survive for several weeks and differentiate into osteoclasts in response to M-CSF and RANKL. Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D(3) analogue given to those mice. Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice. These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.

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Effects of RANKL and M-CSF on the incorporation of BrdU into nuclei of osteoclasts. (A and B) Administration of RANKL to RANKL−/− mice. 3-wk-old RANKL−/− mice were i.p. injected with RANKL (15 µg/injection/day) together with BrdU (1 mg/injection/day) for 2 d. The first injection of BrdU was performed 3 h before the first injection of RANKL. 24 h after the final injection, the tibiae were recovered. (A) Sections of tibiae were prepared and double stained for TRAP (red) and BrdU (brown). Portions of the epiphyseal growth plate (middle) and trabecular bone (right) were observed at a higher magnification. (top) Osteoclasts were totally absent in RANKL−/− mice. (bottom) The administration of RANKL to RANKL−/− mice induced osteoclasts to form in trabecular bones. (B, left) BrdU+ and BrdU− nuclei of chondrocytes in growth plates were counted, and percentages of BrdU+ nuclei in chondrocytes were calculated. (right) BrdU+ and BrdU− nuclei of RANKL-induced osteoclasts were counted, and percentages of BrdU+ nuclei in the osteoclasts were calculated. (C and D) Administration of M-CSF to op/op mice. 3-wk-old op/op mice were i.p. injected with M-CSF (2 × 106 U/injection/day) together with BrdU (1 mg/injection/day) daily for 7 d. The first injection of BrdU was performed 3 h before the first injection of M-CSF. 24 h after the final injection, the tibiae were removed from the mice. (C) Sections of tibiae were prepared and double stained for TRAP (red) and BrdU (brown). Portions of epiphyseal growth plate (middle) and trabecular bone (right) were observed at a higher magnification. (top) Osteoclasts were totally absent in the tibiae of 3-wk-old op/op mice. (bottom) The administration of M-CSF to op/op mice induced osteoclasts to form in trabecular bones. (D, left) BrdU+ and BrdU− nuclei of chondrocytes in growth plates were counted and percentages of BrdU+ nuclei in chondrocytes were calculated. (right) BrdU+ and BrdU− nuclei of M-CSF–induced osteoclasts were counted, and percentages of BrdU+ nuclei in the osteoclasts were calculated. The results are expressed as the mean ± SD for three animals.
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fig4: Effects of RANKL and M-CSF on the incorporation of BrdU into nuclei of osteoclasts. (A and B) Administration of RANKL to RANKL−/− mice. 3-wk-old RANKL−/− mice were i.p. injected with RANKL (15 µg/injection/day) together with BrdU (1 mg/injection/day) for 2 d. The first injection of BrdU was performed 3 h before the first injection of RANKL. 24 h after the final injection, the tibiae were recovered. (A) Sections of tibiae were prepared and double stained for TRAP (red) and BrdU (brown). Portions of the epiphyseal growth plate (middle) and trabecular bone (right) were observed at a higher magnification. (top) Osteoclasts were totally absent in RANKL−/− mice. (bottom) The administration of RANKL to RANKL−/− mice induced osteoclasts to form in trabecular bones. (B, left) BrdU+ and BrdU− nuclei of chondrocytes in growth plates were counted, and percentages of BrdU+ nuclei in chondrocytes were calculated. (right) BrdU+ and BrdU− nuclei of RANKL-induced osteoclasts were counted, and percentages of BrdU+ nuclei in the osteoclasts were calculated. (C and D) Administration of M-CSF to op/op mice. 3-wk-old op/op mice were i.p. injected with M-CSF (2 × 106 U/injection/day) together with BrdU (1 mg/injection/day) daily for 7 d. The first injection of BrdU was performed 3 h before the first injection of M-CSF. 24 h after the final injection, the tibiae were removed from the mice. (C) Sections of tibiae were prepared and double stained for TRAP (red) and BrdU (brown). Portions of epiphyseal growth plate (middle) and trabecular bone (right) were observed at a higher magnification. (top) Osteoclasts were totally absent in the tibiae of 3-wk-old op/op mice. (bottom) The administration of M-CSF to op/op mice induced osteoclasts to form in trabecular bones. (D, left) BrdU+ and BrdU− nuclei of chondrocytes in growth plates were counted and percentages of BrdU+ nuclei in chondrocytes were calculated. (right) BrdU+ and BrdU− nuclei of M-CSF–induced osteoclasts were counted, and percentages of BrdU+ nuclei in the osteoclasts were calculated. The results are expressed as the mean ± SD for three animals.

Mentions: We next examined whether osteoclasts are formed from cell cycle–arrested QuOPs in response to several stimuli. Osteoclasts are totally absent in RANKL−/− mice. Injection of RANKL into RANKL−/− mice induces osteoclasts along bone surfaces (Yamamoto et al., 2006). We first examined whether RANKL-induced osteoclasts are formed from QuOPs in RANKL−/− mice. RANKL (15 µg/injection) was administered i.p. into 3-wk-old RANKL−/− mice for 2 d (one injection/day; Fig. 4, A and B). BrdU was also i.p. injected into RANKL−/− mice (1 mg/injection/day) because toothless RANKL−/− mice were maintained with a water-containing paste diet. Osteoclasts were totally absent in tibiae of RANKL−/− mice (Fig. 4 A, top). The injection of RANKL into RANKL−/− mice generated many TRAP+ cells in tibiae (Fig. 4 A, bottom). BrdU+ nuclei were similarly observed in chondrocytes in growth plates in RANKL−/− mice injected with RANKL (Fig. 4, A [middle] and B [left]). Although many multinucleated osteoclasts were generated by the injection, >70% of the nuclei in the osteoclasts did not incorporate BrdU (Fig. 4, A and B, right). This suggests that RANKL induced the differentiation of preexisting QuOPs in bone tissues into osteoclasts in RANKL−/− mice.


Identification of cell cycle-arrested quiescent osteoclast precursors in vivo.

Mizoguchi T, Muto A, Udagawa N, Arai A, Yamashita T, Hosoya A, Ninomiya T, Nakamura H, Yamamoto Y, Kinugawa S, Nakamura M, Nakamichi Y, Kobayashi Y, Nagasawa S, Oda K, Tanaka H, Tagaya M, Penninger JM, Ito M, Takahashi N - J. Cell Biol. (2009)

Effects of RANKL and M-CSF on the incorporation of BrdU into nuclei of osteoclasts. (A and B) Administration of RANKL to RANKL−/− mice. 3-wk-old RANKL−/− mice were i.p. injected with RANKL (15 µg/injection/day) together with BrdU (1 mg/injection/day) for 2 d. The first injection of BrdU was performed 3 h before the first injection of RANKL. 24 h after the final injection, the tibiae were recovered. (A) Sections of tibiae were prepared and double stained for TRAP (red) and BrdU (brown). Portions of the epiphyseal growth plate (middle) and trabecular bone (right) were observed at a higher magnification. (top) Osteoclasts were totally absent in RANKL−/− mice. (bottom) The administration of RANKL to RANKL−/− mice induced osteoclasts to form in trabecular bones. (B, left) BrdU+ and BrdU− nuclei of chondrocytes in growth plates were counted, and percentages of BrdU+ nuclei in chondrocytes were calculated. (right) BrdU+ and BrdU− nuclei of RANKL-induced osteoclasts were counted, and percentages of BrdU+ nuclei in the osteoclasts were calculated. (C and D) Administration of M-CSF to op/op mice. 3-wk-old op/op mice were i.p. injected with M-CSF (2 × 106 U/injection/day) together with BrdU (1 mg/injection/day) daily for 7 d. The first injection of BrdU was performed 3 h before the first injection of M-CSF. 24 h after the final injection, the tibiae were removed from the mice. (C) Sections of tibiae were prepared and double stained for TRAP (red) and BrdU (brown). Portions of epiphyseal growth plate (middle) and trabecular bone (right) were observed at a higher magnification. (top) Osteoclasts were totally absent in the tibiae of 3-wk-old op/op mice. (bottom) The administration of M-CSF to op/op mice induced osteoclasts to form in trabecular bones. (D, left) BrdU+ and BrdU− nuclei of chondrocytes in growth plates were counted and percentages of BrdU+ nuclei in chondrocytes were calculated. (right) BrdU+ and BrdU− nuclei of M-CSF–induced osteoclasts were counted, and percentages of BrdU+ nuclei in the osteoclasts were calculated. The results are expressed as the mean ± SD for three animals.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654120&req=5

fig4: Effects of RANKL and M-CSF on the incorporation of BrdU into nuclei of osteoclasts. (A and B) Administration of RANKL to RANKL−/− mice. 3-wk-old RANKL−/− mice were i.p. injected with RANKL (15 µg/injection/day) together with BrdU (1 mg/injection/day) for 2 d. The first injection of BrdU was performed 3 h before the first injection of RANKL. 24 h after the final injection, the tibiae were recovered. (A) Sections of tibiae were prepared and double stained for TRAP (red) and BrdU (brown). Portions of the epiphyseal growth plate (middle) and trabecular bone (right) were observed at a higher magnification. (top) Osteoclasts were totally absent in RANKL−/− mice. (bottom) The administration of RANKL to RANKL−/− mice induced osteoclasts to form in trabecular bones. (B, left) BrdU+ and BrdU− nuclei of chondrocytes in growth plates were counted, and percentages of BrdU+ nuclei in chondrocytes were calculated. (right) BrdU+ and BrdU− nuclei of RANKL-induced osteoclasts were counted, and percentages of BrdU+ nuclei in the osteoclasts were calculated. (C and D) Administration of M-CSF to op/op mice. 3-wk-old op/op mice were i.p. injected with M-CSF (2 × 106 U/injection/day) together with BrdU (1 mg/injection/day) daily for 7 d. The first injection of BrdU was performed 3 h before the first injection of M-CSF. 24 h after the final injection, the tibiae were removed from the mice. (C) Sections of tibiae were prepared and double stained for TRAP (red) and BrdU (brown). Portions of epiphyseal growth plate (middle) and trabecular bone (right) were observed at a higher magnification. (top) Osteoclasts were totally absent in the tibiae of 3-wk-old op/op mice. (bottom) The administration of M-CSF to op/op mice induced osteoclasts to form in trabecular bones. (D, left) BrdU+ and BrdU− nuclei of chondrocytes in growth plates were counted and percentages of BrdU+ nuclei in chondrocytes were calculated. (right) BrdU+ and BrdU− nuclei of M-CSF–induced osteoclasts were counted, and percentages of BrdU+ nuclei in the osteoclasts were calculated. The results are expressed as the mean ± SD for three animals.
Mentions: We next examined whether osteoclasts are formed from cell cycle–arrested QuOPs in response to several stimuli. Osteoclasts are totally absent in RANKL−/− mice. Injection of RANKL into RANKL−/− mice induces osteoclasts along bone surfaces (Yamamoto et al., 2006). We first examined whether RANKL-induced osteoclasts are formed from QuOPs in RANKL−/− mice. RANKL (15 µg/injection) was administered i.p. into 3-wk-old RANKL−/− mice for 2 d (one injection/day; Fig. 4, A and B). BrdU was also i.p. injected into RANKL−/− mice (1 mg/injection/day) because toothless RANKL−/− mice were maintained with a water-containing paste diet. Osteoclasts were totally absent in tibiae of RANKL−/− mice (Fig. 4 A, top). The injection of RANKL into RANKL−/− mice generated many TRAP+ cells in tibiae (Fig. 4 A, bottom). BrdU+ nuclei were similarly observed in chondrocytes in growth plates in RANKL−/− mice injected with RANKL (Fig. 4, A [middle] and B [left]). Although many multinucleated osteoclasts were generated by the injection, >70% of the nuclei in the osteoclasts did not incorporate BrdU (Fig. 4, A and B, right). This suggests that RANKL induced the differentiation of preexisting QuOPs in bone tissues into osteoclasts in RANKL−/− mice.

Bottom Line: Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D(3) analogue given to those mice.Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice.These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Oral Science, Matsumoto Dental University, Nagano 399-0781, Japan.

ABSTRACT
Osteoclasts are multinucleated cells that resorb bone. Although osteoclasts originate from the monocyte/macrophage lineage, osteoclast precursors are not well characterized in vivo. The relationship between proliferation and differentiation of osteoclast precursors is examined in this study using murine macrophage cultures treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB (RANK) ligand (RANKL). Cell cycle-arrested quiescent osteoclast precursors (QuOPs) were identified as the committed osteoclast precursors in vitro. In vivo experiments show that QuOPs survive for several weeks and differentiate into osteoclasts in response to M-CSF and RANKL. Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D(3) analogue given to those mice. Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice. These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.

Show MeSH
Related in: MedlinePlus