Proteomic analysis of osteoclast lipid rafts: the role of the integrity of lipid rafts on V-ATPase activity in osteoclasts

Lipid rafts are membrane platforms for signaling molecules that regulate various cellular functions. During osteoclastogenesis, lipid rafts also have been shown to play a crucial role for cell fusion upon RANK/RANKL interaction and further activation of osteoclasts, resulting in bone resorption. To investigate proteins that localize in lipid rafts, we conducted two-dimensional (2D) gel electrophoresis followed by MALDI-TOF mass spectrometry. We identified 12 functional proteins among 34 proteins that were detectable by silver staining of the 2D gel. Among them, a subunit of vacuolar H+-ATPase that is involved in resorption activity of osteoclasts was identified showing an approximate molecular weight of 56.94 kDa and pI of 5.4. The solubilization of osteoclast membrane proteins followed by sucrose density gradient fractionation confirmed that V-ATPase colocalized with flotillin-1, a marker of lipid rafts, in low-density detergent-insoluble fractions. Disruption of rafts with cholesterol-sequestering agents or introduction of a dominant-negative mutant of TRAF6 impaired V-ATPase activity. These data indicate that integrity of lipid rafts regulates the activity of V-ATPase in osteoclasts, suggesting that cholesterol-lowering agents might be useful for inhibiting osteoclast-dependent bone resorption.