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Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors.

Arai F, Miyamoto T, Ohneda O, Inada T, Sudo T, Brasel K, Miyata T, Anderson DM, Suda T - J. Exp. Med. (1999)

Bottom Line: However, how their precursor cells diverge from macrophagic lineages is not known.We have identified early and late stages of osteoclastogenesis, in which precursor cells sequentially express c-Fms followed by receptor activator of nuclear factor kappaB (RANK), and have demonstrated that RANK expression in early-stage of precursor cells (c-Fms(+)RANK(-)) was stimulated by macrophage colony-stimulating factor (M-CSF).Thus, the RANK-RANKL system determines the osteoclast differentiation of bipotential precursors in the default pathway of macrophagic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Differentiation, Institute of Molecular Embryology, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan.

ABSTRACT
Osteoclasts are terminally differentiated cells derived from hematopoietic stem cells. However, how their precursor cells diverge from macrophagic lineages is not known. We have identified early and late stages of osteoclastogenesis, in which precursor cells sequentially express c-Fms followed by receptor activator of nuclear factor kappaB (RANK), and have demonstrated that RANK expression in early-stage of precursor cells (c-Fms(+)RANK(-)) was stimulated by macrophage colony-stimulating factor (M-CSF). Although M-CSF and RANKL (ligand) induced commitment of late-stage precursor cells (c-Fms(+)RANK(+)) into osteoclasts, even late-stage precursors have the potential to differentiate into macrophages without RANKL. Pretreatment of precursors with M-CSF and delayed addition of RANKL showed that timing of RANK expression and subsequent binding of RANKL are critical for osteoclastogenesis. Thus, the RANK-RANKL system determines the osteoclast differentiation of bipotential precursors in the default pathway of macrophagic differentiation.

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Differentiation of c-Kit+Mac-1dullc-Fms− cells. Cells derived from R5 and R6 fractions were cultured for 2 d with SCF (100 ng/ml; cultured cells were designated R5′ and R6′, respectively). (A) Expression of c-Fms on R5′ and R6′ cells. A gate was set on c-Fms+ cells, and the expression of c-Kit was analyzed. A fluorescence histogram shows the c-Kit staining profile of the fraction gated with c-Fms+. (B) Expression of Mac-1 and c-Fms on c-Kit+ cells. (C) c-Fms+ cells were sorted from R5′ or R6′. 103 or 2.5 × 102 cells of each fraction were cocultured with ST2 stromal cells and 1,25-(OH)2D3 (10−8 M) for 4 d, and TRAP activity was measured. (D) Limiting dilution analysis of unfractionated BM mononuclear cells (▵), R3 (•), R5 (▪), and c-Kit+c-Fms+ R5′ cells (□). Cells were cocultured with ST2 stromal cells for 4 d, and the percentages of TRAP+ cells were determined.
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Figure 2: Differentiation of c-Kit+Mac-1dullc-Fms− cells. Cells derived from R5 and R6 fractions were cultured for 2 d with SCF (100 ng/ml; cultured cells were designated R5′ and R6′, respectively). (A) Expression of c-Fms on R5′ and R6′ cells. A gate was set on c-Fms+ cells, and the expression of c-Kit was analyzed. A fluorescence histogram shows the c-Kit staining profile of the fraction gated with c-Fms+. (B) Expression of Mac-1 and c-Fms on c-Kit+ cells. (C) c-Fms+ cells were sorted from R5′ or R6′. 103 or 2.5 × 102 cells of each fraction were cocultured with ST2 stromal cells and 1,25-(OH)2D3 (10−8 M) for 4 d, and TRAP activity was measured. (D) Limiting dilution analysis of unfractionated BM mononuclear cells (▵), R3 (•), R5 (▪), and c-Kit+c-Fms+ R5′ cells (□). Cells were cocultured with ST2 stromal cells for 4 d, and the percentages of TRAP+ cells were determined.

Mentions: To investigate whether R5 cells (c-Kit+Mac-1dullc-Fms−) differentiate to R3 cells (c-Kit+Mac-1dullc-Fms+), the expression of c-Fms was analyzed after cultivation (Fig. 2 A). c-Fms− cells in R5 or R6 were sorted and cultured in SCF (100 ng/ml), as they expressed c-Kit receptors. After 2 d in culture, 42.2% of cultured R5 cells (R5′) expressed c-Fms and 7.1% of c-Fms+ cells were also c-Kit+. In contrast, of cultured R6 cells (R6′), 9.3% were c-Fms+ cells and 0.4% were c-Kit+c-Fms+ cells. Moreover, of R5′ cells, c-Kit+ c-Fms+ cells were mainly Mac-1dull (80.3%; Fig. 2 B). To determine if c-Fms+ cells in R5′ or R6′ could undergo osteoclastic differentiation, both cell fractions were cocultured with ST2 stromal cells for 4 d in the presence of both 1,25-(OH)2D3 and Dex and assayed for TRAP activity (Fig. 2 C). Although c-Fms+ cells of R5′ differentiated to osteoclasts, c-Fms+ cells of R6′ did not differentiate into TRAP+ cells. R6′ cells were mature granulocytes and macrophages.


Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors.

Arai F, Miyamoto T, Ohneda O, Inada T, Sudo T, Brasel K, Miyata T, Anderson DM, Suda T - J. Exp. Med. (1999)

Differentiation of c-Kit+Mac-1dullc-Fms− cells. Cells derived from R5 and R6 fractions were cultured for 2 d with SCF (100 ng/ml; cultured cells were designated R5′ and R6′, respectively). (A) Expression of c-Fms on R5′ and R6′ cells. A gate was set on c-Fms+ cells, and the expression of c-Kit was analyzed. A fluorescence histogram shows the c-Kit staining profile of the fraction gated with c-Fms+. (B) Expression of Mac-1 and c-Fms on c-Kit+ cells. (C) c-Fms+ cells were sorted from R5′ or R6′. 103 or 2.5 × 102 cells of each fraction were cocultured with ST2 stromal cells and 1,25-(OH)2D3 (10−8 M) for 4 d, and TRAP activity was measured. (D) Limiting dilution analysis of unfractionated BM mononuclear cells (▵), R3 (•), R5 (▪), and c-Kit+c-Fms+ R5′ cells (□). Cells were cocultured with ST2 stromal cells for 4 d, and the percentages of TRAP+ cells were determined.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195707&req=5

Figure 2: Differentiation of c-Kit+Mac-1dullc-Fms− cells. Cells derived from R5 and R6 fractions were cultured for 2 d with SCF (100 ng/ml; cultured cells were designated R5′ and R6′, respectively). (A) Expression of c-Fms on R5′ and R6′ cells. A gate was set on c-Fms+ cells, and the expression of c-Kit was analyzed. A fluorescence histogram shows the c-Kit staining profile of the fraction gated with c-Fms+. (B) Expression of Mac-1 and c-Fms on c-Kit+ cells. (C) c-Fms+ cells were sorted from R5′ or R6′. 103 or 2.5 × 102 cells of each fraction were cocultured with ST2 stromal cells and 1,25-(OH)2D3 (10−8 M) for 4 d, and TRAP activity was measured. (D) Limiting dilution analysis of unfractionated BM mononuclear cells (▵), R3 (•), R5 (▪), and c-Kit+c-Fms+ R5′ cells (□). Cells were cocultured with ST2 stromal cells for 4 d, and the percentages of TRAP+ cells were determined.
Mentions: To investigate whether R5 cells (c-Kit+Mac-1dullc-Fms−) differentiate to R3 cells (c-Kit+Mac-1dullc-Fms+), the expression of c-Fms was analyzed after cultivation (Fig. 2 A). c-Fms− cells in R5 or R6 were sorted and cultured in SCF (100 ng/ml), as they expressed c-Kit receptors. After 2 d in culture, 42.2% of cultured R5 cells (R5′) expressed c-Fms and 7.1% of c-Fms+ cells were also c-Kit+. In contrast, of cultured R6 cells (R6′), 9.3% were c-Fms+ cells and 0.4% were c-Kit+c-Fms+ cells. Moreover, of R5′ cells, c-Kit+ c-Fms+ cells were mainly Mac-1dull (80.3%; Fig. 2 B). To determine if c-Fms+ cells in R5′ or R6′ could undergo osteoclastic differentiation, both cell fractions were cocultured with ST2 stromal cells for 4 d in the presence of both 1,25-(OH)2D3 and Dex and assayed for TRAP activity (Fig. 2 C). Although c-Fms+ cells of R5′ differentiated to osteoclasts, c-Fms+ cells of R6′ did not differentiate into TRAP+ cells. R6′ cells were mature granulocytes and macrophages.

Bottom Line: However, how their precursor cells diverge from macrophagic lineages is not known.We have identified early and late stages of osteoclastogenesis, in which precursor cells sequentially express c-Fms followed by receptor activator of nuclear factor kappaB (RANK), and have demonstrated that RANK expression in early-stage of precursor cells (c-Fms(+)RANK(-)) was stimulated by macrophage colony-stimulating factor (M-CSF).Thus, the RANK-RANKL system determines the osteoclast differentiation of bipotential precursors in the default pathway of macrophagic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Differentiation, Institute of Molecular Embryology, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan.

ABSTRACT
Osteoclasts are terminally differentiated cells derived from hematopoietic stem cells. However, how their precursor cells diverge from macrophagic lineages is not known. We have identified early and late stages of osteoclastogenesis, in which precursor cells sequentially express c-Fms followed by receptor activator of nuclear factor kappaB (RANK), and have demonstrated that RANK expression in early-stage of precursor cells (c-Fms(+)RANK(-)) was stimulated by macrophage colony-stimulating factor (M-CSF). Although M-CSF and RANKL (ligand) induced commitment of late-stage precursor cells (c-Fms(+)RANK(+)) into osteoclasts, even late-stage precursors have the potential to differentiate into macrophages without RANKL. Pretreatment of precursors with M-CSF and delayed addition of RANKL showed that timing of RANK expression and subsequent binding of RANKL are critical for osteoclastogenesis. Thus, the RANK-RANKL system determines the osteoclast differentiation of bipotential precursors in the default pathway of macrophagic differentiation.

Show MeSH
Related in: MedlinePlus