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The role of the small Rho GTPase Rac2 in mature osteoclasts has not been extensively studied. Rac2−/− mice are of normal size and have normal tooth eruption. However, femoral cortical thickness was significantly greater in Rac2−/− compared to wild-type mice, while percent cortical porosity was lower. As assessed by histomorphometry, trabecular bone mass was significantly higher in male Rac2−/− than wild-type animals, although trabecular bone mass was similar when data from male and female animals were combined. There were no significant differences in the number of osteoblasts per bone surface; however, the number of osteoclasts per total bone area tended to be higher in Rac2−/− mice and was significantly higher in male Rac2−/− mice. In the aggregate, these data suggested a defect in osteoclast function and, consistent with that, rates of bone resorption were significantly reduced in Rac2−/− osteoclasts. In addition, Rac2−/− osteoclasts had a significantly delayed spreading response to treatment with CSF1 for 15 min. Phalloidin staining showed areas of abnormal actin accumulation and impaired actin ring formation in Rac2−/− osteoclasts. Finally, Rac2−/− osteoclasts showed a marked defect in chemotaxis toward a point source of CSF1, with a dramatic reduction in migratory rate. Together, these findings indicate an important role for Rac2 in mature osteoclasts.

The cytoskeleton determines cell shape and is involved in cell motility. It also plays a role in differentiation and in modulating specialized cellular functions. LIM kinase 1 (LIMK1) participates in cytoskeletal remodeling by phosphorylating and inactivating the actin-severing protein, cofilin. Severing F-actin to release G-actin monomers is required for actin cytoskeletal remodeling. Although less well established, LIMK1 may also influence the cell cycle and modulate metalloproteinase activity. Since the role of LIMK1 in bone cell biology has not been reported, the skeletal phenotype of LIMK1−/− mice was examined. LIMK1−/− mice had significantly reduced trabecular bone mass when analyzed by micoCT (p <0.01). Histomorphometric analyses demonstrated a 31% reduction in the number osteoblasts (p=0.0003) and a 23% reduction in osteoid surface (p=0.0005). The number of osteoclasts was no different in control and knock out animals. Consistent with the in vivo findings in osteoblasts, the number of osteoblast colony forming units in LIMK1−/− bone marrow was reduced by nearly 50%. Further, osteoblasts isolated from LIMK1−/− mice showed significantly reduced rates of mineralization in vitro. Osteoclasts from LIMK1−/− mice evidenced more rapid cytoskeletal remodeling in response to treatment with CSF1. In keeping with this latter finding, basal levels of phospho-cofilin were reduced in LIMK1−/− osteoclasts. LIMK1−/− osteoclasts also resorbed dentine slices to a greater extent in vitro and were more active in a pit assay. These data support the hypothesis that LIMK1 is required for normal osteoblast differentiation. In addition, its absence leads to increased cytoskeletal remodeling and bone resorption in osteoclasts.

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.

Antibody-mediated blockade of cluster of differentiation 47 (CD47)-thrombospondin-1 (TSP-1) interactions blocks osteoclast formation and attenuates parathyroid hormone (PTH)-induced hypercalcemia in mice. Hypercalcemia in this model reflects increased bone resorption. TSP-1 has two cell-associated binding partners, CD47 and CD36. The roles of these two molecules in mediating the effects of TSP1 in osteoclasts are unclear. Osteoclast formation was attenuated but not absent when preosteoclasts isolated from CD47 mice were cocultured with WT osteoblasts. Suppressing CD36 in osteoclast progenitors also attenuated osteoclast formation. The hypercalcemic response to a PTH infusion was blunted in CD47/CD36 (double knockout (DKO)) female mice but not CD47 mice or CD36 animals, supporting a role for both CD47 and CD36 in mediating this effect. Consistent with this, DKO osteoclasts had impaired resorptive activity when analyzed Inhibition of nitric oxide (NO) signaling is known to promote osteoclastogenesis, and TSP-1 suppresses NO production and signaling. An anti-TSP-1 antibody increased NO production in osteoclasts, and the inhibitory effect of anti-TSP-1 on osteoclastogenesis was completely rescued by l-nitroarginine methyl ester (l-NAME), a competitive NO synthase inhibitor. Supportive of an important role for CD36 in mediating the pro-osteoclastogenic effects of TSP-1, engaging CD36 with a synthetic agonist, p907, suppressed NO production in anti-TSP-1-treated cultures, allowing osteoclast maturation to occur. These results establish that CD36 and CD47 both participate in mediating the actions of TSP-1 in osteoclasts and establish a physiologically relevant cross-talk in bone tissue between these two molecules.

Vascular remodeling is a characteristic feature of cardiovascular diseases. Altered cellular processes of vascular smooth muscle cells (VSMCs) is a crucial component in vascular remodeling. Histone deacetylase inhibitor (HDACI), butyrate, arrests VSMC proliferation and promotes cell growth. The objective of the study is to determine the mechanism of butyrate-induced VSMC growth. Using proliferating VSMCs exposed to 5 mM butyrate, immunoblotting studies are performed to determine whether PI3K/Akt pathway that regulates different cellular effects is a target of butyrate-induced VSMC growth. Butyrate inhibits phosphorylation-dependent activation of PI3K, PDK1, and Akt, eliciting differential effects on downstream targets of Akt. Along with previously reported Ser9 phosphorylation-mediated GSK3 inactivation leading to stability, increased expression and accumulation of cyclin D1, and epigenetic histone modifications, inactivation of Akt by butyrate results in: transcriptional activation of FOXO1 and FOXO3 promoting G1 arrest through p21Cip1/Waf1 and p15INK4B upregulation; inactivation of mTOR inhibiting activation of its targets p70S6K and 4E-BP1 impeding protein synthesis; inhibition of caspase 3 cleavage and downregulation of PARP preventing apoptosis. Our findings imply butyrate abrogates Akt activation, causing differential effects on Akt targets promoting convergence of cross-talk between their complimentary actions leading to VSMC growth by arresting proliferation and inhibiting apoptosis through its effect on dual targets, HDAC activity and PI3K/Akt pathway network.

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