1183 The Wiscott-Aldrich and Scar Homolog (WASH) complex in osteoclasts

Saturday, March 24, 2012: 8 a.m. - 9:30 a.m.
Presentation Type: Oral Session
L. HOLLIDAY, E. TORO, and J. ZUO, Orthodontics, University of Florida, Gainesville, FL
Objectives: Several lines of evidence suggest that binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments is vital for osteoclast function.  Recent data show that enoxacin, a small molecule inhibitor of this interaction, inhibits osteoclast bone resorption in vitro, and both cancer growth and metastasis and horizontal alveolar bone loss triggered by polymicrobial periodontal infections in rodent model systems.  Binding between V-ATPase and microfilaments has been shown to be vital for vesicle sorting controlled by the WASH complex. Here we tested whether the WASH complex is involved in osteoclast function

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.

This abstract is based on research that was funded entirely or partially by an outside source: R21 DE19862 T32 DE07200-15

Keywords: Cell biology, Osteoblasts/osteoclasts, Periodontal disease, Remodeling and Resorption