Suppression of RANKL-induced osteoclast differentiation by cilostazol via SIRT1-induced RANK inhibition

• Regulation of osteoclastogenesis provides a potential strategy for treatment of bone damage.
• Cilostazol elevates SIRT1 mRNA and protein levels and increases SIRT1 activity.
• RANKL-induced RANK expression is attenuated by cilostazol and rSIRT1.
• RANKL-induced TRAP+ giant cells and resorption pit formation are inhibited by cilostazol.
• Cilostazol inhibits RANKL-induced osteoclastogenesis.

Osteoclasts are bone-specific multinucleated cells generated by differentiation of monocyte/macrophage hematopoietic lineages and degrade bone matrix by secretion of lytic enzymes. The regulation of osteoclast differentiation provides a potential strategy for treatment of bone-lytic damage. In this study, cilostazol, an inhibitor of type III phosphodiesterase, inhibited RANKL [receptor activator of nuclear factor kappa B (RANK) ligand]-induced RANK expression in bone marrow-derived monocyte/macrophage precursors (BMMs) and Raw 264.7 cells by inhibiting PU.1 via SIRT1 activation. RANKL-induced RANK expression was attenuated by cilostazol and rSIRT1 in Raw 264.7 cells, and these were blocked by sirtinol. In line with these, cilostazol elevated SIRT1 mRNA and protein levels in 12–24 h and increased SIRT1 activity, and these effects were inhibited by sirtinol. Furthermore, the RANKL-induced nuclear expression of PU.1, a transcription factor required for macrophage differentiation, was suppressed by cilostazol. Additionally, marked RANKL-induced RANK immunofluorescence staining in Raw 264.7 cells was attenuated by cilostazol and rSIRT1, and both attenuations were prevented by sirtinol. Extensive RANK staining of knee synovial tissues in a mouse model of collagen-induced arthritis (CIA) was markedly reduced by cilostazol (30 mg/kg/day). In line with these results, both RANKL- and M-CSF-induced differentiation of BMMs to multinucleated TRAP+ giant cells and resorption pit formation were inhibited by cilostazol associated with a decrease in TRAP (a marker enzyme of osteoclasts) activity. In conclusion, cilostazol activates SIRT1, which suppresses the nuclear translocation of PU.1, and thus, inhibits RANKL-stimulated RANK expression and causes anti-osteoclast formation in BMMs in vitro and in their murine model of CIA.