FOXO1 mediates RANKL-induced osteoclast formation and activity.

Wang Y, Dong G, Jeon HH, Elazizi M, La LB, Hameedaldeen A, Xiao E, Tian C, Alsadun S, Choi Y, Graves DT.
Source: J Immunol
Publication Date: (2015)
Issue: 194(6): 2878-87
Research Area:
Dermatology/Tissue Engineering
Basic Research
Cells used in publication:
Macrophage, mouse
Species: mouse
Tissue Origin: bone marrow
RAW 264.7
Species: mouse
Tissue Origin: blood

Osteoclast bone resorption activity was measured with the OsteoLyse assay kit (Lonza, Allendale, NJ) according to the manufacturer’s protocol. Briefly, BMMs from experimental and control mice or RAW264.7 cells transfected with FOXO1 siRNA or control scrambled siRNA were cultured in 96-well OsteoLyse plates (Lonza) at 50,000 cells/well in complete a-MEM. BMMs were incubated with 30 ng/ml M-CSF and 100 ng/ml soluble RANKL, and RAW265.7 cells incubated with 100 ng/ml soluble RANKL. Four days later culture medium was renewed and samples were collected after an additional 24 h of culture, and calcium release was measured by fluorescence.


We have previously shown that the transcription factor FOXO1 is elevated in conditions with high levels of bone resorption. To investigate the role of FOXO1 in the formation of osteoclasts, we examined mice with lineage-specific deletion of FOXO1 in osteoclast precursors and by knockdown of FOXO1 with small interfering RNA. The receptor activator for NF-?B ligand (RANKL), a principal bone-resorbing factor, induced FOXO1 expression and nuclear localization 2 d after stimulation in bone marrow macrophages and RAW264.7 osteoclast precursors. RANKL-induced osteoclast formation and osteoclast activity was reduced in half in vivo and in vitro with lineage-specific FOXO1 deletion (LyzM.Cre(+)FOXO1(L/L)) compared with matched controls (LyzM.Cre(-)FOXO1(L/L)). Similar results were obtained by knockdown of FOXO1 in RAW264.7 cells. Moreover, FOXO1-mediated osteoclast formation was linked to regulation of NFATc1 nuclear localization and expression as well as a number of downstream factors, including dendritic cell-specific transmembrane protein, ATP6vod2, cathepsin K, and integrin av. Lastly, FOXO1 deletion reduced M-CSF-induced RANK expression and migration of osteoclast precursors. In the present study, we provide evidence that FOXO1 plays a direct role in osteoclast formation by mediating the effect of RANKL on NFATc1 and several downstream effectors. This is likely to be significant because FOXO1 and RANKL are elevated in osteolytic conditions.