Characterization of Rac GTPase Function in the Osteoclast

Osteoclasts are the unique mediator of bone resorption in the body. Balancing bone resorption with osteoblast mediated bone matrix formation is essential for maintaining a healthy and structurally strong skeleton. When the balanced actions of these two cell types are impaired, disease results. The most common bone disease in the United States is osteoporosis, the pathological loss of bone that results in fragility and increased fracture risk. Understanding the molecular pathways that control osteoclast differentiation and function is requisite in determining targets of therapeutic intervention. Actin plays a key role in mediating osteoclast function. Not only must the osteoclast migrate to sites of bone resorption, it also organizes the unique actin sealing zone to initiate cell polarization and bone resorption. Chemicals that block actin remodeling and organization abolish osteoclast mediated bone resorption in vitro. The Rho family of small GTPases is well described as regulating the actin cytoskeleton. Rho is classically associated with stress fiber formation, Cdc42 with filopodia formation and Rac with membrane ruffles and lamellipodia. We sought to determine the impact of Rac GTPase activity on osteoclast differentiation and function. We hypothesized that Rac mediates the organization of the osteoclast actin ring and cytoskeleton. Using a genetic model of Rac isoform deletion in the mouse, we find that while loss of Rac1 or Rac 2, alone, has no effect, deletion of Rac1 in osteoclastic cells of Rac2-/- mice yields severe osteopetrosis. Osteoclasts lacking Rac1 and 2 (RacDKO), in combination, fail to effectively resorb bone. On the other hand, osteoclasts are abundant in RacDKO osteopetrotic mice and normally express the cells\u27 maturation markers. Hence, the osteopetrotic lesion of RacDKO mice largely reflects impaired function and not arrested differentiation of the resorptive polykaryon. The dysfunction of RacDKO osteoclasts represents failed cytoskeleton organization as evidenced by reduced motility and inability to spread or generate the key resorptive organelles, actin rings and ruffled borders, accompanied by abnormal Arp3 distribution. The cytoskeleton-organizing capacity of Rac1 is mediated via its 20 amino acid effector domain. Thus, Rac1 and Rac2 are mutually compensatory. Unlike cdc42 deficiency, their combined absence does not impact differentiation but promotes severe osteopetrosis by dysregulating the osteoclast cytoskeleton. Through this study we conclude that Rac GTPase activity is a key modulator of the osteoclast cytoskeleton and is required for sealing zone formation and osteoclast function