Rac-GTPase, osteoclast cytoskeleton and bone resorption

The members of the Rho-GTPase subfamily, Rad and Rad, are intimately involv ed in the organization of the cytoskeleton, and the p21-activated kinases o r PAKs are targets of these proteins. Rad and Rac2 are also essential compo nents of NADPH oxidase, the enzyme responsible for generating free radicals . The cytoskeleton modulates the adhesion of osteoclasts to bone and its su bsequent resorption. These cells contain NADPH diaphorase activity, and fre e radicals influence bone resorption. The influence of Rac1, Rac2 and PAK1 on the cytoskeleton, resorbing activity and NADPH diaphorase activity of di saggregated rat osteoclasts was investigated by permeabilisation with sapon in and introducing specific anti-Rad, anti-Rac2 or anti-PAK1 antibodies. Rh odamine-phalloidin stain was used to identify actin in osteoclasts cultured on plastic slides, and the bone-slice method was used to measure resorptio n. Saponin permeabilisation did not affect the cytoskeletal organization or bone resorption. Anti-Rac antibodies caused dose- and time-dependent cytos keletal changes. The osteoclasts rounded up and developed retraction fibers ; actin rings were disrupted and large actin dots were seen at the peripher y of the cells. Osteoclast resorptive activity was depressed after incubati on with the antibodies. The total area resorbed by treated cells and the me an pit area were smaller than those of controls. Anti-PAK1 antibody caused similar changes. None of the antibodies altered the NADPH diaphorase activi ty. Thus, Rac-GTPases are present in rat osteoclasts and are involved in th e organization of the actin cytoskeleton and in resorptive activity. These effects may be mediated by PAK1 kinase, but do not influence osteoclast NAD PH diaphorase activity.