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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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Localization of QuOPs in bone. (A) Localization of c-Fms+/RANK+ and Ki67+ cells. Tibiae were recovered from 7-wk-old wild-type and 3-wk-old RANKL−/− mice. Sections of tibiae were prepared and subjected to double staining of RANK (green) and c-Fms (red; top and middle). Nuclei were labeled with DAPI (blue). Top panels show low power views of the specimens, and middle and bottom panels show high power views. (middle) The asterisk indicates a multinucleated osteoclast, which is surrounded by a small dotted line, and arrows indicate mononuclear cells double positive for RANK and c-Fms (yellow). Tibiae were also recovered from 7-wk-old wild-type mice pretreated with 5-FU for 6 d. (bottom left) Sections of tibiae from 5-FU–treated mice were stained for RANK (green), c-Fms (red), and DAPI (blue). The arrow indicates cells double positive for c-Fms and RANK in 5-FU–treated mice. (bottom right) Sections of tibiae from RANKL−/− mice were also stained for RANK (green), Ki67 (red), and DAPI (blue). The arrow indicates a RANK+ and Ki67− cell. Bones are indicated by dashed lines. (B) Localization of RANK+ cells and ALP+ cells. Sections of tibiae from RANKL−/− mice were subjected to staining of RANK (green), ALP (red), and DAPI (blue). Arrows and arrowheads indicate RANK+ and ALP+ cells, respectively. The inset shows a high power view of the boxed region.
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fig7: Localization of QuOPs in bone. (A) Localization of c-Fms+/RANK+ and Ki67+ cells. Tibiae were recovered from 7-wk-old wild-type and 3-wk-old RANKL−/− mice. Sections of tibiae were prepared and subjected to double staining of RANK (green) and c-Fms (red; top and middle). Nuclei were labeled with DAPI (blue). Top panels show low power views of the specimens, and middle and bottom panels show high power views. (middle) The asterisk indicates a multinucleated osteoclast, which is surrounded by a small dotted line, and arrows indicate mononuclear cells double positive for RANK and c-Fms (yellow). Tibiae were also recovered from 7-wk-old wild-type mice pretreated with 5-FU for 6 d. (bottom left) Sections of tibiae from 5-FU–treated mice were stained for RANK (green), c-Fms (red), and DAPI (blue). The arrow indicates cells double positive for c-Fms and RANK in 5-FU–treated mice. (bottom right) Sections of tibiae from RANKL−/− mice were also stained for RANK (green), Ki67 (red), and DAPI (blue). The arrow indicates a RANK+ and Ki67− cell. Bones are indicated by dashed lines. (B) Localization of RANK+ cells and ALP+ cells. Sections of tibiae from RANKL−/− mice were subjected to staining of RANK (green), ALP (red), and DAPI (blue). Arrows and arrowheads indicate RANK+ and ALP+ cells, respectively. The inset shows a high power view of the boxed region.

Mentions: Finally, we tried to detect c-Fms+/RANK+ cells in bone tissues in RANKL−/− mice because QuOPs but not osteoclasts are present in RANKL−/− mice (Fig. 4, A and B). Many mononuclear cells double positive for c-Fms and RANK (c-Fms+/RANK+ cells; Fig. 7 A, yellow) were detected along the surface of proximal tibiae in wild-type mice (Fig. 7 A, left, arrows). Double-positive multinucleated cells were also observed in the same regions (Fig. 7 A, asterisk). These multinucleated cells appeared to be osteoclasts. QuOPs were shown to be resistant to 5-FU treatment (Figs. 5 and 6). Coincidently, c-Fms+/RANK+ mononuclear cells were also detected even after treatment with 5-FU (Fig. 7 A, bottom left). c-Fms+/RANK+ mononuclear cells were detected along the surface of proximal tibiae in the RANKL−/− mice (Fig. 7 A, right). Because most RANK+ cells expressed c-Fms in RANKL−/− mice, we examined the expression of Ki67, a marker of actively cycling cells. Most of the RANK+ cells (94%) were negative for Ki67 in RANKL−/− mice (Fig. 7 A, bottom right).


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)

Localization of QuOPs in bone. (A) Localization of c-Fms+/RANK+ and Ki67+ cells. Tibiae were recovered from 7-wk-old wild-type and 3-wk-old RANKL−/− mice. Sections of tibiae were prepared and subjected to double staining of RANK (green) and c-Fms (red; top and middle). Nuclei were labeled with DAPI (blue). Top panels show low power views of the specimens, and middle and bottom panels show high power views. (middle) The asterisk indicates a multinucleated osteoclast, which is surrounded by a small dotted line, and arrows indicate mononuclear cells double positive for RANK and c-Fms (yellow). Tibiae were also recovered from 7-wk-old wild-type mice pretreated with 5-FU for 6 d. (bottom left) Sections of tibiae from 5-FU–treated mice were stained for RANK (green), c-Fms (red), and DAPI (blue). The arrow indicates cells double positive for c-Fms and RANK in 5-FU–treated mice. (bottom right) Sections of tibiae from RANKL−/− mice were also stained for RANK (green), Ki67 (red), and DAPI (blue). The arrow indicates a RANK+ and Ki67− cell. Bones are indicated by dashed lines. (B) Localization of RANK+ cells and ALP+ cells. Sections of tibiae from RANKL−/− mice were subjected to staining of RANK (green), ALP (red), and DAPI (blue). Arrows and arrowheads indicate RANK+ and ALP+ cells, respectively. The inset shows a high power view of the boxed region.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: Localization of QuOPs in bone. (A) Localization of c-Fms+/RANK+ and Ki67+ cells. Tibiae were recovered from 7-wk-old wild-type and 3-wk-old RANKL−/− mice. Sections of tibiae were prepared and subjected to double staining of RANK (green) and c-Fms (red; top and middle). Nuclei were labeled with DAPI (blue). Top panels show low power views of the specimens, and middle and bottom panels show high power views. (middle) The asterisk indicates a multinucleated osteoclast, which is surrounded by a small dotted line, and arrows indicate mononuclear cells double positive for RANK and c-Fms (yellow). Tibiae were also recovered from 7-wk-old wild-type mice pretreated with 5-FU for 6 d. (bottom left) Sections of tibiae from 5-FU–treated mice were stained for RANK (green), c-Fms (red), and DAPI (blue). The arrow indicates cells double positive for c-Fms and RANK in 5-FU–treated mice. (bottom right) Sections of tibiae from RANKL−/− mice were also stained for RANK (green), Ki67 (red), and DAPI (blue). The arrow indicates a RANK+ and Ki67− cell. Bones are indicated by dashed lines. (B) Localization of RANK+ cells and ALP+ cells. Sections of tibiae from RANKL−/− mice were subjected to staining of RANK (green), ALP (red), and DAPI (blue). Arrows and arrowheads indicate RANK+ and ALP+ cells, respectively. The inset shows a high power view of the boxed region.
Mentions: Finally, we tried to detect c-Fms+/RANK+ cells in bone tissues in RANKL−/− mice because QuOPs but not osteoclasts are present in RANKL−/− mice (Fig. 4, A and B). Many mononuclear cells double positive for c-Fms and RANK (c-Fms+/RANK+ cells; Fig. 7 A, yellow) were detected along the surface of proximal tibiae in wild-type mice (Fig. 7 A, left, arrows). Double-positive multinucleated cells were also observed in the same regions (Fig. 7 A, asterisk). These multinucleated cells appeared to be osteoclasts. QuOPs were shown to be resistant to 5-FU treatment (Figs. 5 and 6). Coincidently, c-Fms+/RANK+ mononuclear cells were also detected even after treatment with 5-FU (Fig. 7 A, bottom left). c-Fms+/RANK+ mononuclear cells were detected along the surface of proximal tibiae in the RANKL−/− mice (Fig. 7 A, right). Because most RANK+ cells expressed c-Fms in RANKL−/− mice, we examined the expression of Ki67, a marker of actively cycling cells. Most of the RANK+ cells (94%) were negative for Ki67 in RANKL−/− mice (Fig. 7 A, bottom right).

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