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FOXO1 inhibits osteoclastogenesis partially by antagnozing MYC.

Tan P, Guan H, Xie L, Mi B, Fang Z, Li J, Li F - Sci Rep (2015)

Bottom Line: Intriguingly, recent studies on the role played by FOXOs in osteoclastogenesis reached different conclusion.We found that FOXO1 inhibition modulated MAPKs, NF-κB and AP-1.To conclude, our study confirmed FOXO1 as a cell-autonomous inhibitor of osteoclastogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.

ABSTRACT
FOXO transcription factors especially FOXO1 have profound roles in bone development and remodeling. The regulation of cells of the osteoblast lineage by FOXOs is suggested to be stage-specific or context dependent. Intriguingly, recent studies on the role played by FOXOs in osteoclastogenesis reached different conclusion. Bartell et al. showed that FOXOs restrained osteoclastogenesis and bone resorption partially by upregulation of the H2O2-inactivating enzyme catalase. Wang et al. demonstrated that FOXO1 activated osteoclast formation. In the present study, we confirmed the results of Bartell et al. that FOXO1 expression was reduced upon stimulation of RANKL; FOXO1 inhibition promoted and FOXO1 activation repressed, osteoclast differentiation and activity; the inhibitory effect of FOXO1 on osteoclastogenesis was partially mediated by ROS since treatment with ROS scavengers cancelled the effect of FOXO1 inhibition on osteoclastogenesis. We further investigated the mechanisms responsible for repressed osteoclastogenesis by FOXO1. We found that FOXO1 inhibition modulated MAPKs, NF-κB and AP-1. Finally, we proved that the inhibitory effect of FOXO1 on osteoclast formation was partially mediated by MYC suppression by showing that MYC repression almost totally abrogated the effect of FOXO1 inhibition on osteoclastogenesis. To conclude, our study confirmed FOXO1 as a cell-autonomous inhibitor of osteoclastogenesis.

No MeSH data available.


Related in: MedlinePlus

FOXO1 inhibition promoted osteoclast differentiation.(A–C) FOXO1 inhibition promoted osteoclastogenesis of mouse BMMCs. Mouse BMMCs were treated with the indicated doses of FOXO1 inhibitor (AS1842856) and M-CSF (30 ng/ml) and RANKL (50 ng/ml). After 7 days, cells were fixed for TRAP staining (A) and TRAP enzyme activity assay (C). The cells were photographed (original magnification, ×40; A) and the number of TRAP-positive multinucleated (> =3 nuclei) osteoclasts was counted (B). Data represent the mean±SD of 3 independent experiments. (D–F) FOXO1 inhibition promoted osteoclastogenesis of RAW264.7 cells. RAW264.7 cells were incubated with RANKL (50 ng/mL) and the indicated concentrations of AS1842856 for 4 days. TRAP staining and TRAP enzyme activity assay (F) were performed. The cells were photographed (D) and the number of TRAP-positive multinucleated osteoclasts was counted (E). Data represent the mean ± SD of 3 independent experiments. *P < 0.05 versus control group.
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f2: FOXO1 inhibition promoted osteoclast differentiation.(A–C) FOXO1 inhibition promoted osteoclastogenesis of mouse BMMCs. Mouse BMMCs were treated with the indicated doses of FOXO1 inhibitor (AS1842856) and M-CSF (30 ng/ml) and RANKL (50 ng/ml). After 7 days, cells were fixed for TRAP staining (A) and TRAP enzyme activity assay (C). The cells were photographed (original magnification, ×40; A) and the number of TRAP-positive multinucleated (> =3 nuclei) osteoclasts was counted (B). Data represent the mean±SD of 3 independent experiments. (D–F) FOXO1 inhibition promoted osteoclastogenesis of RAW264.7 cells. RAW264.7 cells were incubated with RANKL (50 ng/mL) and the indicated concentrations of AS1842856 for 4 days. TRAP staining and TRAP enzyme activity assay (F) were performed. The cells were photographed (D) and the number of TRAP-positive multinucleated osteoclasts was counted (E). Data represent the mean ± SD of 3 independent experiments. *P < 0.05 versus control group.

Mentions: To explore the roles played by FOXO1 in osteoclastogenesis, we used AS1842856, a specific inhibitor of FOXO1 transcription activity. We first measured the influence of AS1842856 on cell viability. AS1842856 had no significant influence on BMMCs proliferation and slightly promoted the proliferation of RAW264.7 cells (Supplemental Fig. 1). As shown in Fig. 2, treatment with AS1842856 significantly increased the number and size of TRAP-positive multinuclear cells in a dose-dependent manner in both BMMCs and RAW264.7 cells. Similarly, TRAP activity in culture medium also increased following AS1842856 treatment (Fig. 2C,F). The promotion of osteoclastogenesis was confirmed by the higher mRNA levels of TRAP, ATP6v0d2, CK, and MMP9 (Fig. 3A,B). To evaluate the potential influence of FOXO1 inhibition on the resorbing activity of osteoclasts, RAW264.7 cells were induced to differentiate into osteoclasts without AS1842856 treatment. Mature osteoclasts were collected, seeded onto a Corning OsteoAssay Surface and treated with AS1842856 (0.5 μM) in the presence of 50 ng/ml RANKL for 3 days. Then the resorption pits were visualized with light microscopy. As shown in Fig. 3C, FOXO1 inhibition obviously increased the resorption area. When quantified with a software, the increase of the resorption area was 3.4 fold (Fig. 3D).


FOXO1 inhibits osteoclastogenesis partially by antagnozing MYC.

Tan P, Guan H, Xie L, Mi B, Fang Z, Li J, Li F - Sci Rep (2015)

FOXO1 inhibition promoted osteoclast differentiation.(A–C) FOXO1 inhibition promoted osteoclastogenesis of mouse BMMCs. Mouse BMMCs were treated with the indicated doses of FOXO1 inhibitor (AS1842856) and M-CSF (30 ng/ml) and RANKL (50 ng/ml). After 7 days, cells were fixed for TRAP staining (A) and TRAP enzyme activity assay (C). The cells were photographed (original magnification, ×40; A) and the number of TRAP-positive multinucleated (> =3 nuclei) osteoclasts was counted (B). Data represent the mean±SD of 3 independent experiments. (D–F) FOXO1 inhibition promoted osteoclastogenesis of RAW264.7 cells. RAW264.7 cells were incubated with RANKL (50 ng/mL) and the indicated concentrations of AS1842856 for 4 days. TRAP staining and TRAP enzyme activity assay (F) were performed. The cells were photographed (D) and the number of TRAP-positive multinucleated osteoclasts was counted (E). Data represent the mean ± SD of 3 independent experiments. *P < 0.05 versus control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4645183&req=5

f2: FOXO1 inhibition promoted osteoclast differentiation.(A–C) FOXO1 inhibition promoted osteoclastogenesis of mouse BMMCs. Mouse BMMCs were treated with the indicated doses of FOXO1 inhibitor (AS1842856) and M-CSF (30 ng/ml) and RANKL (50 ng/ml). After 7 days, cells were fixed for TRAP staining (A) and TRAP enzyme activity assay (C). The cells were photographed (original magnification, ×40; A) and the number of TRAP-positive multinucleated (> =3 nuclei) osteoclasts was counted (B). Data represent the mean±SD of 3 independent experiments. (D–F) FOXO1 inhibition promoted osteoclastogenesis of RAW264.7 cells. RAW264.7 cells were incubated with RANKL (50 ng/mL) and the indicated concentrations of AS1842856 for 4 days. TRAP staining and TRAP enzyme activity assay (F) were performed. The cells were photographed (D) and the number of TRAP-positive multinucleated osteoclasts was counted (E). Data represent the mean ± SD of 3 independent experiments. *P < 0.05 versus control group.
Mentions: To explore the roles played by FOXO1 in osteoclastogenesis, we used AS1842856, a specific inhibitor of FOXO1 transcription activity. We first measured the influence of AS1842856 on cell viability. AS1842856 had no significant influence on BMMCs proliferation and slightly promoted the proliferation of RAW264.7 cells (Supplemental Fig. 1). As shown in Fig. 2, treatment with AS1842856 significantly increased the number and size of TRAP-positive multinuclear cells in a dose-dependent manner in both BMMCs and RAW264.7 cells. Similarly, TRAP activity in culture medium also increased following AS1842856 treatment (Fig. 2C,F). The promotion of osteoclastogenesis was confirmed by the higher mRNA levels of TRAP, ATP6v0d2, CK, and MMP9 (Fig. 3A,B). To evaluate the potential influence of FOXO1 inhibition on the resorbing activity of osteoclasts, RAW264.7 cells were induced to differentiate into osteoclasts without AS1842856 treatment. Mature osteoclasts were collected, seeded onto a Corning OsteoAssay Surface and treated with AS1842856 (0.5 μM) in the presence of 50 ng/ml RANKL for 3 days. Then the resorption pits were visualized with light microscopy. As shown in Fig. 3C, FOXO1 inhibition obviously increased the resorption area. When quantified with a software, the increase of the resorption area was 3.4 fold (Fig. 3D).

Bottom Line: Intriguingly, recent studies on the role played by FOXOs in osteoclastogenesis reached different conclusion.We found that FOXO1 inhibition modulated MAPKs, NF-κB and AP-1.To conclude, our study confirmed FOXO1 as a cell-autonomous inhibitor of osteoclastogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.

ABSTRACT
FOXO transcription factors especially FOXO1 have profound roles in bone development and remodeling. The regulation of cells of the osteoblast lineage by FOXOs is suggested to be stage-specific or context dependent. Intriguingly, recent studies on the role played by FOXOs in osteoclastogenesis reached different conclusion. Bartell et al. showed that FOXOs restrained osteoclastogenesis and bone resorption partially by upregulation of the H2O2-inactivating enzyme catalase. Wang et al. demonstrated that FOXO1 activated osteoclast formation. In the present study, we confirmed the results of Bartell et al. that FOXO1 expression was reduced upon stimulation of RANKL; FOXO1 inhibition promoted and FOXO1 activation repressed, osteoclast differentiation and activity; the inhibitory effect of FOXO1 on osteoclastogenesis was partially mediated by ROS since treatment with ROS scavengers cancelled the effect of FOXO1 inhibition on osteoclastogenesis. We further investigated the mechanisms responsible for repressed osteoclastogenesis by FOXO1. We found that FOXO1 inhibition modulated MAPKs, NF-κB and AP-1. Finally, we proved that the inhibitory effect of FOXO1 on osteoclast formation was partially mediated by MYC suppression by showing that MYC repression almost totally abrogated the effect of FOXO1 inhibition on osteoclastogenesis. To conclude, our study confirmed FOXO1 as a cell-autonomous inhibitor of osteoclastogenesis.

No MeSH data available.


Related in: MedlinePlus