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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 5-FU on changes in hemopoietic cell populations. (A) Flow cytometric analysis of bone marrow cells. 7-wk-old mice were injected with vehicle (top) or 5-FU (250 mg/kg body weight; bottom). 8 d after the injection, mice were killed. Bone marrow cells prepared from tibiae were analyzed for the expression of MOMA-2 and CD11b (left) and c-Fms and RANK (right). The numbers in the top right corners indicate percentages of MOMA-2+/CD11b+ cells or of c-Fms+/RANK+ cells among all bone marrow cells. Results are expressed as the mean ± SD for three animals. (B) Effects of HU on osteoclast formation. MOMA-2+/CD11b+ cells and c-Fms+/RANK+ cells were isolated from bone marrow of wild-type mice by magnetic cell sorting and cultured in the presence of 104 U/ml M-CSF and 100 ng/ml RANKL. 70 µM HU was added to some cultures. After 6 d of culture, cells were fixed and stained for TRAP. TRAP+ multinucleated cells containing more than three nuclei were counted as osteoclasts. Results are expressed as the mean ± SD for six cultures. *, P < 0.01; significantly different from the culture incubated without HU.
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fig6: Effects of 5-FU on changes in hemopoietic cell populations. (A) Flow cytometric analysis of bone marrow cells. 7-wk-old mice were injected with vehicle (top) or 5-FU (250 mg/kg body weight; bottom). 8 d after the injection, mice were killed. Bone marrow cells prepared from tibiae were analyzed for the expression of MOMA-2 and CD11b (left) and c-Fms and RANK (right). The numbers in the top right corners indicate percentages of MOMA-2+/CD11b+ cells or of c-Fms+/RANK+ cells among all bone marrow cells. Results are expressed as the mean ± SD for three animals. (B) Effects of HU on osteoclast formation. MOMA-2+/CD11b+ cells and c-Fms+/RANK+ cells were isolated from bone marrow of wild-type mice by magnetic cell sorting and cultured in the presence of 104 U/ml M-CSF and 100 ng/ml RANKL. 70 µM HU was added to some cultures. After 6 d of culture, cells were fixed and stained for TRAP. TRAP+ multinucleated cells containing more than three nuclei were counted as osteoclasts. Results are expressed as the mean ± SD for six cultures. *, P < 0.01; significantly different from the culture incubated without HU.

Mentions: The aforementioned data suggest that QuOPs are resistant to 5-FU and are present in bone. QuOPs as well as osteoclasts are expected to express both M-CSF receptors (c-Fms) and RANKL receptors (RANK; Miyamoto et al., 2000). Next, we examined whether the administration of 5-FU alters the population of c-Fms+/RANK+ cells (c-Fms and RANK double-positive cells) in the bone marrow. 5-FU was i.v. injected into 7-wk-old mice. On day 8 after injection, bone marrow cells were collected and analyzed with a flow cytometer (Fig. 6). MOMA-2+/CD11b+ cells (monocytes/macrophages) decreased in number with the injection (Fig. 6 A, left). In contrast, 5-FU increased the Fms+/RANK+ cell population in the bone marrow cells (Fig. 6 A, right), suggesting that c-Fms+/RANK+ cells were cell cycle arrested. We then examined whether MOMA-2+/CD11b+ cells and c-Fms+/RANK+ cells can differentiate into osteoclasts without proliferating. MOMA-2+/CD11b+ cells differentiated into TRAP+ osteoclasts in response to M-CSF and RANKL in the absence but not presence of HU (Fig. 6 B). In contrast, c-Fms+/RANK+ cells differentiated into TRAP+ cells even in the presence of HU (Fig. 6 B).


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 5-FU on changes in hemopoietic cell populations. (A) Flow cytometric analysis of bone marrow cells. 7-wk-old mice were injected with vehicle (top) or 5-FU (250 mg/kg body weight; bottom). 8 d after the injection, mice were killed. Bone marrow cells prepared from tibiae were analyzed for the expression of MOMA-2 and CD11b (left) and c-Fms and RANK (right). The numbers in the top right corners indicate percentages of MOMA-2+/CD11b+ cells or of c-Fms+/RANK+ cells among all bone marrow cells. Results are expressed as the mean ± SD for three animals. (B) Effects of HU on osteoclast formation. MOMA-2+/CD11b+ cells and c-Fms+/RANK+ cells were isolated from bone marrow of wild-type mice by magnetic cell sorting and cultured in the presence of 104 U/ml M-CSF and 100 ng/ml RANKL. 70 µM HU was added to some cultures. After 6 d of culture, cells were fixed and stained for TRAP. TRAP+ multinucleated cells containing more than three nuclei were counted as osteoclasts. Results are expressed as the mean ± SD for six cultures. *, P < 0.01; significantly different from the culture incubated without HU.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Effects of 5-FU on changes in hemopoietic cell populations. (A) Flow cytometric analysis of bone marrow cells. 7-wk-old mice were injected with vehicle (top) or 5-FU (250 mg/kg body weight; bottom). 8 d after the injection, mice were killed. Bone marrow cells prepared from tibiae were analyzed for the expression of MOMA-2 and CD11b (left) and c-Fms and RANK (right). The numbers in the top right corners indicate percentages of MOMA-2+/CD11b+ cells or of c-Fms+/RANK+ cells among all bone marrow cells. Results are expressed as the mean ± SD for three animals. (B) Effects of HU on osteoclast formation. MOMA-2+/CD11b+ cells and c-Fms+/RANK+ cells were isolated from bone marrow of wild-type mice by magnetic cell sorting and cultured in the presence of 104 U/ml M-CSF and 100 ng/ml RANKL. 70 µM HU was added to some cultures. After 6 d of culture, cells were fixed and stained for TRAP. TRAP+ multinucleated cells containing more than three nuclei were counted as osteoclasts. Results are expressed as the mean ± SD for six cultures. *, P < 0.01; significantly different from the culture incubated without HU.
Mentions: The aforementioned data suggest that QuOPs are resistant to 5-FU and are present in bone. QuOPs as well as osteoclasts are expected to express both M-CSF receptors (c-Fms) and RANKL receptors (RANK; Miyamoto et al., 2000). Next, we examined whether the administration of 5-FU alters the population of c-Fms+/RANK+ cells (c-Fms and RANK double-positive cells) in the bone marrow. 5-FU was i.v. injected into 7-wk-old mice. On day 8 after injection, bone marrow cells were collected and analyzed with a flow cytometer (Fig. 6). MOMA-2+/CD11b+ cells (monocytes/macrophages) decreased in number with the injection (Fig. 6 A, left). In contrast, 5-FU increased the Fms+/RANK+ cell population in the bone marrow cells (Fig. 6 A, right), suggesting that c-Fms+/RANK+ cells were cell cycle arrested. We then examined whether MOMA-2+/CD11b+ cells and c-Fms+/RANK+ cells can differentiate into osteoclasts without proliferating. MOMA-2+/CD11b+ cells differentiated into TRAP+ osteoclasts in response to M-CSF and RANKL in the absence but not presence of HU (Fig. 6 B). In contrast, c-Fms+/RANK+ cells differentiated into TRAP+ cells even in the presence of HU (Fig. 6 B).

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