Deletion of Rac in Mature Osteoclasts Causes Osteopetrosis, an Age-Dependent Change in Osteoclast Number, and a Reduced Number of Osteoblasts In Vivo

Abstract

Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1flox/flox mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2−/− mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism.

LRRK1 regulation of actin assembly in osteoclasts involves serine 5 phosphorylation of L‐plastin

“…We also noted that the integrin αvβ3, c-Src, and Rac/cdc42 small GTPase proteins are key regulators of the OC cytoskeleton and activity (24-27) . Deletion of any of these signaling molecules compromises the capacity of OCs to remodel the cytoskeleton and resorb bone, leading to osteopetrosis phenotypes.…”

Mouse Cre Models for the Study of Bone Diseases

“…Conditional gene deletion can be especially informative in situations where global deletion of the gene is embryonic lethal (which is particularly problematical for bone research if lethality occurs prior to formation of the skeletal elements) or where postnatal lethality prevents researchers from examining the function of the gene in the postnatal or adult skeleton. Bone-specific Cre mouse models have also provided a powerful tool for dissecting out the precise function of genes in specific bone cell lineages [1–5]. Additionally, Cre models have been used with great success for lineage tracing studies in mineralized tissues [6–8].…”

Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation

“…While Rac1/2-deficient osteoclasts in vitro and Rac1/2 KO mice exhibited bone resorption defects, the magnitude of phenotypic changes caused by lack of Rac1/2 is less severe than that of Lrrk1-deficient cells or Lrrk1 KO mice. Furthermore, mice with deletion of Rac1/2 in mature osteoclasts also had a normal response to PTH treatment, just as in the case of Lrrk1 KO mice (27, 38). In addition, RAC1 has been reported to interact with Lrrk2 protein (6).…”

“…Here, we demonstrated that PKC substrates with molecular masses of ∼110, 57–78, and 27 kDa were phosphorylated at significantly lower levels in Lrrk1 KO osteoclasts compared with WT cells. While several signaling pathways including integrin, NF-κB, and Src could be involved in regulating osteoclast function (14), we focused on small GTPase Rac/Cdc42 signaling pathways since previous studies showed that double KO of Rac1 and Rac2 in mice caused osteopetrosis due to cytoskeleton disarrangement and osteoclast dysfunction (8, 38). While Rac1/2-deficient osteoclasts in vitro and Rac1/2 KO mice exhibited bone resorption defects, the magnitude of phenotypic changes caused by lack of Rac1/2 is less severe than that of Lrrk1-deficient cells or Lrrk1 KO mice.…”