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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 TRAP+ cells from RANK+ or RANK− cells. R3 cells were precultured with M-CSF (30 ng/ml) for 24 or 72 h. These precultured cells and primary R3 cells were then divided into RANK+ or RANK− fractions. Primary and cultured RANK+ or RANK− cells were cultured with sRANKL (25 ng/ml) and M-CSF (100 ng/ml). (A and B) Primary R3 cells. (C and D) 24-h M-CSF–precultured cells. (E and F) M-CSF 72-h–precultured cells. TRAP staining (A, C, and E) was performed on day 2 (I and IV), day 4 (II and V), or day 6 (III and VI), and the percentage of TRAP+ cells or TRAP+ MNCs (B, D, and F) was scored at the same time. I, II, and III were derived from RANK+ cells; IV, V, and VI were from RANK− cells. Scale bar, 100 μm. Upper panels of B, D, and F represent percentages of TRAP+ cells that include both mononuclear cells and MNCs in the total cells in the well. Lower panels of B, D, and F represent percentages of TRAP+ MNCs in total TRAP+ cells in the well. Percent of TRAP+ cells or TRAP+ MNCs derived from RANK+ (▪) or RANK− (□) cells is shown.
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Figure 6: Differentiation of TRAP+ cells from RANK+ or RANK− cells. R3 cells were precultured with M-CSF (30 ng/ml) for 24 or 72 h. These precultured cells and primary R3 cells were then divided into RANK+ or RANK− fractions. Primary and cultured RANK+ or RANK− cells were cultured with sRANKL (25 ng/ml) and M-CSF (100 ng/ml). (A and B) Primary R3 cells. (C and D) 24-h M-CSF–precultured cells. (E and F) M-CSF 72-h–precultured cells. TRAP staining (A, C, and E) was performed on day 2 (I and IV), day 4 (II and V), or day 6 (III and VI), and the percentage of TRAP+ cells or TRAP+ MNCs (B, D, and F) was scored at the same time. I, II, and III were derived from RANK+ cells; IV, V, and VI were from RANK− cells. Scale bar, 100 μm. Upper panels of B, D, and F represent percentages of TRAP+ cells that include both mononuclear cells and MNCs in the total cells in the well. Lower panels of B, D, and F represent percentages of TRAP+ MNCs in total TRAP+ cells in the well. Percent of TRAP+ cells or TRAP+ MNCs derived from RANK+ (▪) or RANK− (□) cells is shown.

Mentions: To clarify the synergistic effect of M-CSF and sRANKL, the relationship between the onset of RANK expression and osteoclast differentiation was examined. Fig. 5 shows the flow chart of cell sorting and conditions of further cultivation. Sorted RANK+ or RANK− cells from the primary R3 fraction were cultured for 2, 4, and 6 d with both sRANKL and M-CSF (Fig. 6A and Fig. B). On days 2 and 4, the percentage of TRAP+ cells was higher in RANK− cells (11.8 ± 1.9% on day 2 and 97.5 ± 1.4% on day 4) than in RANK+ cells (7.0 ± 1.7% on day 2 and 42.5 ± 6.1% on day 4). On day 6, however, the percentage of TRAP+ cells was similar in RANK+ and RANK− cells. Interestingly, the percentage of TRAP+ cells in R3 cells precultured for 24 h with M-CSF was similar between RANK+ and RANK− cells (Fig. 6C and Fig. D), whereas in the case of R3 cells that were not precultured, RANK+ cells did not efficiently differentiate into TRAP+ cells (Fig. 6A and Fig. B). RANK− cells showed a higher percentage of MNCs, which are fully matured osteoclasts, than did RANK+ cells. These MNCs from RANK− cells were extremely large and contained a large number of nuclei.


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 TRAP+ cells from RANK+ or RANK− cells. R3 cells were precultured with M-CSF (30 ng/ml) for 24 or 72 h. These precultured cells and primary R3 cells were then divided into RANK+ or RANK− fractions. Primary and cultured RANK+ or RANK− cells were cultured with sRANKL (25 ng/ml) and M-CSF (100 ng/ml). (A and B) Primary R3 cells. (C and D) 24-h M-CSF–precultured cells. (E and F) M-CSF 72-h–precultured cells. TRAP staining (A, C, and E) was performed on day 2 (I and IV), day 4 (II and V), or day 6 (III and VI), and the percentage of TRAP+ cells or TRAP+ MNCs (B, D, and F) was scored at the same time. I, II, and III were derived from RANK+ cells; IV, V, and VI were from RANK− cells. Scale bar, 100 μm. Upper panels of B, D, and F represent percentages of TRAP+ cells that include both mononuclear cells and MNCs in the total cells in the well. Lower panels of B, D, and F represent percentages of TRAP+ MNCs in total TRAP+ cells in the well. Percent of TRAP+ cells or TRAP+ MNCs derived from RANK+ (▪) or RANK− (□) cells is shown.
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Figure 6: Differentiation of TRAP+ cells from RANK+ or RANK− cells. R3 cells were precultured with M-CSF (30 ng/ml) for 24 or 72 h. These precultured cells and primary R3 cells were then divided into RANK+ or RANK− fractions. Primary and cultured RANK+ or RANK− cells were cultured with sRANKL (25 ng/ml) and M-CSF (100 ng/ml). (A and B) Primary R3 cells. (C and D) 24-h M-CSF–precultured cells. (E and F) M-CSF 72-h–precultured cells. TRAP staining (A, C, and E) was performed on day 2 (I and IV), day 4 (II and V), or day 6 (III and VI), and the percentage of TRAP+ cells or TRAP+ MNCs (B, D, and F) was scored at the same time. I, II, and III were derived from RANK+ cells; IV, V, and VI were from RANK− cells. Scale bar, 100 μm. Upper panels of B, D, and F represent percentages of TRAP+ cells that include both mononuclear cells and MNCs in the total cells in the well. Lower panels of B, D, and F represent percentages of TRAP+ MNCs in total TRAP+ cells in the well. Percent of TRAP+ cells or TRAP+ MNCs derived from RANK+ (▪) or RANK− (□) cells is shown.
Mentions: To clarify the synergistic effect of M-CSF and sRANKL, the relationship between the onset of RANK expression and osteoclast differentiation was examined. Fig. 5 shows the flow chart of cell sorting and conditions of further cultivation. Sorted RANK+ or RANK− cells from the primary R3 fraction were cultured for 2, 4, and 6 d with both sRANKL and M-CSF (Fig. 6A and Fig. B). On days 2 and 4, the percentage of TRAP+ cells was higher in RANK− cells (11.8 ± 1.9% on day 2 and 97.5 ± 1.4% on day 4) than in RANK+ cells (7.0 ± 1.7% on day 2 and 42.5 ± 6.1% on day 4). On day 6, however, the percentage of TRAP+ cells was similar in RANK+ and RANK− cells. Interestingly, the percentage of TRAP+ cells in R3 cells precultured for 24 h with M-CSF was similar between RANK+ and RANK− cells (Fig. 6C and Fig. D), whereas in the case of R3 cells that were not precultured, RANK+ cells did not efficiently differentiate into TRAP+ cells (Fig. 6A and Fig. B). RANK− cells showed a higher percentage of MNCs, which are fully matured osteoclasts, than did RANK+ cells. These MNCs from RANK− cells were extremely large and contained a large number of nuclei.

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