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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 might promote osteoclastogenesis through upregulation of MYC expression.(A,B) RAW264.7 cells were treated with the indicated doses of AS1842856. 24 hours later, cells were harvested and the mRNA level of MYC was assessed by Q-PCR (A). Data represent the mean ± SD of 3 independent experiments. 48 hours after treatment, cells were collected and the protein level of MYC was detected by Immunoblotting (B). (C,D) RAW264.7 cells were incubated with RANKL (50 ng/mL), followed by treatment with the indicated doses of FOXO1 inhibitor (AS1842856) and MYC inhibitor (10058-F4). Four days later, cells were fixed for TRAP staining. The cells were photographed (original magnification,×40) and the numbers of TRAP-positive multinucleated (> = 3 nuclei) osteoclasts were counted (E,F). *P < 0.05 versus control group, #P < 0.05 versus AS1842856 0.5 μM group.
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f7: FOXO1 inhibition might promote osteoclastogenesis through upregulation of MYC expression.(A,B) RAW264.7 cells were treated with the indicated doses of AS1842856. 24 hours later, cells were harvested and the mRNA level of MYC was assessed by Q-PCR (A). Data represent the mean ± SD of 3 independent experiments. 48 hours after treatment, cells were collected and the protein level of MYC was detected by Immunoblotting (B). (C,D) RAW264.7 cells were incubated with RANKL (50 ng/mL), followed by treatment with the indicated doses of FOXO1 inhibitor (AS1842856) and MYC inhibitor (10058-F4). Four days later, cells were fixed for TRAP staining. The cells were photographed (original magnification,×40) and the numbers of TRAP-positive multinucleated (> = 3 nuclei) osteoclasts were counted (E,F). *P < 0.05 versus control group, #P < 0.05 versus AS1842856 0.5 μM group.

Mentions: MYC is a transcription factor involved in multiple cellular processes and is required for osteoclast differentiation27. We have recently shown that FOXO1 activation almost completely abolished MYC protein expression in lymphoma cells17. We speculated MYC might have mediated the effect of FOXO1 on osteoclastogenesis. MYC expression after FOXO1 inhibition was measured by Q-PCR and western blot, as shown in Fig. 7A,B, FOXO1 inhibition promoted the expression of MYC at the mRNA and especially the protein level. To further elucidate the role of MYC in FOXO1 mediated repression of osteoclastogenesis, we used 10058-F4, a small molecule inhibitor of MYC. RAW264.7 cells were treated with both AS1842856 and 10058-F4 in the presence of RANKL (50 ng/ml). 4 days later, cells were fixed for TRAP staining and images were taken. 10058-F4 abrogated the pro-osteoclastogenesis effect of FOXO1 inhibition in RAW264.7 cells (Fig. 7C,D) in a dose dependent manner.


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 might promote osteoclastogenesis through upregulation of MYC expression.(A,B) RAW264.7 cells were treated with the indicated doses of AS1842856. 24 hours later, cells were harvested and the mRNA level of MYC was assessed by Q-PCR (A). Data represent the mean ± SD of 3 independent experiments. 48 hours after treatment, cells were collected and the protein level of MYC was detected by Immunoblotting (B). (C,D) RAW264.7 cells were incubated with RANKL (50 ng/mL), followed by treatment with the indicated doses of FOXO1 inhibitor (AS1842856) and MYC inhibitor (10058-F4). Four days later, cells were fixed for TRAP staining. The cells were photographed (original magnification,×40) and the numbers of TRAP-positive multinucleated (> = 3 nuclei) osteoclasts were counted (E,F). *P < 0.05 versus control group, #P < 0.05 versus AS1842856 0.5 μM group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: FOXO1 inhibition might promote osteoclastogenesis through upregulation of MYC expression.(A,B) RAW264.7 cells were treated with the indicated doses of AS1842856. 24 hours later, cells were harvested and the mRNA level of MYC was assessed by Q-PCR (A). Data represent the mean ± SD of 3 independent experiments. 48 hours after treatment, cells were collected and the protein level of MYC was detected by Immunoblotting (B). (C,D) RAW264.7 cells were incubated with RANKL (50 ng/mL), followed by treatment with the indicated doses of FOXO1 inhibitor (AS1842856) and MYC inhibitor (10058-F4). Four days later, cells were fixed for TRAP staining. The cells were photographed (original magnification,×40) and the numbers of TRAP-positive multinucleated (> = 3 nuclei) osteoclasts were counted (E,F). *P < 0.05 versus control group, #P < 0.05 versus AS1842856 0.5 μM group.
Mentions: MYC is a transcription factor involved in multiple cellular processes and is required for osteoclast differentiation27. We have recently shown that FOXO1 activation almost completely abolished MYC protein expression in lymphoma cells17. We speculated MYC might have mediated the effect of FOXO1 on osteoclastogenesis. MYC expression after FOXO1 inhibition was measured by Q-PCR and western blot, as shown in Fig. 7A,B, FOXO1 inhibition promoted the expression of MYC at the mRNA and especially the protein level. To further elucidate the role of MYC in FOXO1 mediated repression of osteoclastogenesis, we used 10058-F4, a small molecule inhibitor of MYC. RAW264.7 cells were treated with both AS1842856 and 10058-F4 in the presence of RANKL (50 ng/ml). 4 days later, cells were fixed for TRAP staining and images were taken. 10058-F4 abrogated the pro-osteoclastogenesis effect of FOXO1 inhibition in RAW264.7 cells (Fig. 7C,D) in a dose dependent manner.

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