IADR Abstract Archives
FOXO1 Mediates RANKL Induced Osteoclastogenesis
Objectives: To test the hypothesis that FOXO1 is activated by RANKL and plays a direct role in osteoclast formation stimulated by RANKL.
Methods: LyzM.Cre+FOXO1L/L (experimental) and LyzM.Cre-FOXO1L/L (control) mice were injected with RANKL or vehicle alone in the scalp for 5 consecutive days (n=6 per each group). Pit number and area were measured with microCT-40. Immunofluorescence was carried out with antibodies specific for TRAP, FOXO1, and NFATc1 or non-immune control IgG with DAPI counterstaining. Bone marrow macrophages (BMMs) and RAW264.7 pre-osteoclasts were stimulated with RANKL for 4 days in vitro. The number of multinucleated-TRAP+ cells was measured with a chromogen substrate. Statistical significance was determined by ANOVA with Tukey’s post-hoc test at p<0.05 for multiple comparison or Students t-test for two group comparisons.
Results: LyzM-driven Cre recombinase reduced FOXO1 mRNA levels by 95% and protein levels by 90% in BMM. RANKL stimulated a six fold increase in FOXO1 nuclear localization in osteoclasts compared to vehicle control (p<0.05). This was decreased by 77% in LyzM.Cre+FOXO1L/L(p<0.05). RANKL stimulated 2.1-fold increase in NFATc1+TRAP+ osteoclasts. This was reduced by 70% in LyzM.Cre+FOXO1L/L(p<0.05). Pit number and total pit area stimulated by RANKL were decreased by 60% and 64% respectively in LyzM.Cre+FOXO1L/L compared to control mice (P<0.05). RANKL stimulated osteoclast formation in vivo was reduced by 59% in calvarial sutures and by 70% in bone marrow of LyzM.Cre+FOXO1L/L compared to WT control mice (p<0.05).
In vitro RANKL stimulated a 8-fold FOXO1 increase in BMMs and a 4.5-fold increase in RAW264.7 cells(P<0.05). Increased nuclear localization was detectable two days after RANKL-induction by Western blot. Lineage-specific deletion of FOXO1 reduced the osteoclasts number by 65% in BMMs and 49% in RAW264.7 cells(p<0.05).
Conclusions: 1. RANKL stimulated FOXO1 expression and FOXO1 nuclear localization.
2. FOXO1 deletion significantly reduced osteoclastogenesis stimulated by RANKL in vivo and in vitro.
Methods: LyzM.Cre+FOXO1L/L (experimental) and LyzM.Cre-FOXO1L/L (control) mice were injected with RANKL or vehicle alone in the scalp for 5 consecutive days (n=6 per each group). Pit number and area were measured with microCT-40. Immunofluorescence was carried out with antibodies specific for TRAP, FOXO1, and NFATc1 or non-immune control IgG with DAPI counterstaining. Bone marrow macrophages (BMMs) and RAW264.7 pre-osteoclasts were stimulated with RANKL for 4 days in vitro. The number of multinucleated-TRAP+ cells was measured with a chromogen substrate. Statistical significance was determined by ANOVA with Tukey’s post-hoc test at p<0.05 for multiple comparison or Students t-test for two group comparisons.
Results: LyzM-driven Cre recombinase reduced FOXO1 mRNA levels by 95% and protein levels by 90% in BMM. RANKL stimulated a six fold increase in FOXO1 nuclear localization in osteoclasts compared to vehicle control (p<0.05). This was decreased by 77% in LyzM.Cre+FOXO1L/L(p<0.05). RANKL stimulated 2.1-fold increase in NFATc1+TRAP+ osteoclasts. This was reduced by 70% in LyzM.Cre+FOXO1L/L(p<0.05). Pit number and total pit area stimulated by RANKL were decreased by 60% and 64% respectively in LyzM.Cre+FOXO1L/L compared to control mice (P<0.05). RANKL stimulated osteoclast formation in vivo was reduced by 59% in calvarial sutures and by 70% in bone marrow of LyzM.Cre+FOXO1L/L compared to WT control mice (p<0.05).
In vitro RANKL stimulated a 8-fold FOXO1 increase in BMMs and a 4.5-fold increase in RAW264.7 cells(P<0.05). Increased nuclear localization was detectable two days after RANKL-induction by Western blot. Lineage-specific deletion of FOXO1 reduced the osteoclasts number by 65% in BMMs and 49% in RAW264.7 cells(p<0.05).
Conclusions: 1. RANKL stimulated FOXO1 expression and FOXO1 nuclear localization.
2. FOXO1 deletion significantly reduced osteoclastogenesis stimulated by RANKL in vivo and in vitro.