Background/Purpose:

Dysregulation of osteoclast differentiation and function is a feature of inflammatory arthropathies (IA). Multiple genes and signalling pathways have been associated with regulation of osteoclastogenesis. In particular, FOXO1, a member of the forkhead box O family of transcription factors, is implicated in many cell processes, including metabolism, signalling, ageing, stress, cell cycle, cell differentiation and as such is an intriguing candidate for osteoclast dysregulation in IA. Recent murine studies however provided conflicting data with regard to the role of FOXO1 in osteoclast differentiation (1, 2) and no data are available in human osteoclastogenesis. We therefore investigated the role of FOXO1 in human osteoclast differentiation.

Methods: Human CD14+ monocytes were isolated from human buffy coat samples using magnetic separation kit (Stemcell). Monocytes were differentiated into osteoclasts with M-CSF (25 ng/ml) and RANK-L (25 ng/ml) either in the presence or absence of a highly specific FOXO1 inhibitor (AS1842856; 0.5 uM for 3 days). To mimic inflammation-driven osteoclastogenesis, monocytes were grown in suboptimal RANK-L (1 ng/ml) medium for 4 days and on the 4^th day TNF or supernatants from activated CD3+ T cells, were added in the presence or absence of FOXO1 inhibitor. Mature osteoclasts were stained with tartrate-resistant acid phosphatase and quantified by light microscopy.  Mineral-coated plates were used to assess the resorption activity. Gene expression was assessed by RT-qPCR. 

Results:

Inhibition of FOXO1 completely blocked both RANK-L driven and inflammation-driven (TNF or activated CD3⁺supernatant), osteoclast differentiation of human CD14+ monocytes and strongly inhibited osteolytic activity. FOXO1 inhibited cells were viable and had a macrophage-like phenotype. Kinetic studies revealed that FOXO1 plays a role in both the early cell fate decision and the late commitment phase of osteoclastogenesis; both inhibition during the first three days (pre-osteoclast stage) or later stage (day 4-7; committed osteoclast pre-cursors) was observed. Moreover, selective addition of AS1842856  on only day 1, clearly implicated FOXO1 during the earliest stages of osteoclast differentiation. Finally, gene expression analysis of major osteoclastogenesis-associated genes (M-CSFR, RANK and NFATC1) showed reduction upon treatment with AS1842856.

Conclusion: Our results suggest that FOXO1 is an essential transcription factor for human osteoclast differentiation. The ability to inhibit inflammation-driven osteoclastogenesis and osteolytic function indicates that this pathway may be viable therapeutic target in inflammatory arthropathies.

 References:

1. Wang Y, Dong G, Jeon HH, Elazizi M, La LB, Hameedaldeen A, Xiao E, Tian C, Alsadun S, Choi Y, Graves DT. FOXO1 mediates RANKL-induced osteoclast formation and activity. J Immunol. 2015 Mar 15;194(6):2878-87.
2. Tan P, Guan H, Xie L, Mi B, Fang Z, Li J, Li F. FOXO1 inhibits osteoclastogenesis partially by antagnozing MYC. Sci Rep. 2015 Nov 16;5:16835.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/foxo1-is-required-for-human-osteoclast-differentiation/