Methods: WASH complex proteins were detected by Western blot, real time quantitative PCR and immunofluorescence microscopy, and knocked down from osteoclasts by RNA interference. Osteoclasts were monitored by staining of actin rings with phalloidin and V-ATPase with anti-E subunit antibody.
Results: Elements of the WASH complex, WASH, FAM21 and CapZβ, were upregulated 2-3-fold during osteoclastogenesis. FAM21 and CapZβ were localized to the actin rings of osteoclasts and were associated with vesicles that were enriched in the cytosol above the ruffled plasma membrane of resorbing osteoclasts. Enoxacin disrupts osteoclast actin ring formation. Preliminary knockdown of WASH from RAW 264.7 osteoclast-like cells using siRNA reduced mRNA levels by 65% (by qPCR), and the number of actin rings from 51 ± 6 to 19 ± 11 actin rings/ well of 24 well dishes.
Conclusions: The WASH complex plays an important role in osteoclast function. Enoxacin may exert effects in vivo due to disruption of WASH complex-dependent sorting of V-ATPase.
Keywords: Cell biology, Osteoblasts/osteoclasts, Periodontal disease, Remodeling and Resorption