Inhibition of Osteoclast Activation by Phloretin through Disturbing αvβ3 Integrin-c-Src Pathway

This study was to explore the sequential signaling of disorganization of the actin cytoskeletal architecture by phloretin. RAW 264.7 macrophages were incubated with 1-20 μM phloretin for 5 days in the presence of RANKL. C57BL/6 mice were ovariectomized (OVX) and orally treated with 10 mg/kg phloretin once a day for 8 weeks. Phloretin allayed RANKL stimulated formation of actin podosomes with the concomitant retardation of the vinculin activation. Oral administration of phloretin suppressed the induction of femoral gelsolin and vinculin in OVX mice. The RANK-RANKL interaction resulted in the αvβ3 integrin induction, which was demoted by phloretin. The RANKL induction of actin rings and vacuolar-type H(+)-ATPase entailed Pyk2 phosphorylation and c-Src and c-Cbl induction, all of which were blunted by phloretin. Similar inhibition was also observed in phloretin-exposed OVX mouse femoral bone tissues with decreased trabecular collagen formation. Phloretin suppressed the paxillin induction in RANKL-activated osteoclasts and in OVX epiphyseal bone tissues. Also, phloretin attenuated the Syk phosphorylation and phospholipase Cγ induction by RANKL in osteoclasts. These results suggest that phloretin was an inhibitor of actin podosomes and sealing zone, disrupting αvβ3 integrin-c-Src-Pyk2/Syk signaling pathway for the regulation of actin cytoskeletal organization in osteoclasts.