Sbds is required for Rac2-mediated monocyte migration and signaling downstream of RANK during osteoclastogenesis (original) (raw)
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Src-like adaptor protein regulates osteoclast generation and survival
Journal of Cellular Biochemistry, 2010
Src-like adaptor protein (SLAP) is a hematopoietic adaptor containing Src homology (SH)3 and SH2 motifs and a unique carboxy terminus. Unlike c-Src, SLAP lacks a tyrosine kinase domain. We investigated the role of SLAP in osteoclast development and resorptive function. Employing SLAP-deficient mice, we find lack of the adaptor enhances in vitro proliferation of osteoclast precursors in the form of bone marrow macrophages (BMMs), without altering their survival. Furthermore, osteoclastogenic markers appear more rapidly in SLAP−/− BMMs exposed to RANK ligand (RANKL). The accelerated proliferation of M-CSF-treated, SLAP-deficient precursors is associated with enhanced ERK activation. SLAP's role as a mediator of M-CSF signaling, in osteoclastic cells, is buttressed by complexing of the adaptor protein and c-Fms in lipid rafts. Unlike c-Src, SLAP does not impact resorptive function of mature osteoclasts, but induces their early apoptosis. Thus, SLAP negatively regulates differentiation of osteoclasts and proliferation of their precursors. Conversely, SLAP decreases osteoclast death by inhibiting activation of caspase 3. These counterbalancing events yield indistinguishable bones of WT and SLAP−/− mice which contain equal numbers of osteoclasts in basal and stimulated conditions. Keywords SLAP; osteoclasts; c-Src Multinucleated osteoclasts, the sole bone-resorbing cells, are critical to skeletal development and maintenance [Teitelbaum, 2000]. They are derived from monocyte/macrophage precursors under the aegis of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). While RANKL regulates osteoclast differentiation, M-CSF promotes proliferation of marrow macrophages [Boyle et al., 2003; Pixley and Stanley, 2004; Teitelbaum and Ross, 2003]. RANKL promotes mature osteoclast survival and M-CSF plays the same role for these bone resorbing polykaryons and their precursors [Akiyama et al., 2003; Xaus et al., 2001].
Identifying the Relative Contributions of Rac1 and Rac2 to Osteoclastogenesis
Journal of Bone and Mineral Research, 2007
Rac small GTPases may play an important regulatory role in osteoclastogenesis. Our in vitro and in vivo results show that both Rac1 and Rac2 are required for optimal osteoclast differentiation, but Rac1 is more critical. Rac1 is the key Rac isoform responsible for regulating ROS generation and the actin cytoskeleton during the multiple stages of osteoclast differentiation.
International Journal of Molecular Sciences
Shwachman–Diamond syndrome (SDS) is a rare autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, and skeletal abnormalities, caused by loss-of-function mutations in the SBDS gene, a factor involved in ribosome biogenesis. By analyzing osteoblasts from SDS patients (SDS-OBs), we show that SDS-OBs displayed reduced SBDS gene expression and reduced/undetectable SBDS protein compared to osteoblasts from healthy subjects (H-OBs). SDS-OBs cultured in an osteogenic medium displayed a lower mineralization capacity compared to H-OBs. Whole transcriptome analysis showed significant differences in the gene expression of SDS-OBs vs. H-OBs, particularly in the ossification pathway. SDS-OBs expressed lower levels of the main genes responsible for osteoblastogenesis. Of all downregulated genes, Western blot analyses confirmed lower levels of alkaline phosphatase and collagen type I in SDS-OBs than in H-OBs. Interestingly, SDS-OBs showed higher protei...
The molecular mechanisms that regulate fusion of monocytes into functional osteoclasts are virtually unknown. We describe a knock-in mouse model for the R51Q mutation in sorting nexin 10 (SNX10) that exhibits osteopetrosis and related symptoms of patients of autosomal recessive osteopetrosis linked to this mutation. Osteopetrosis arises in homozygous R51Q SNX10 mice due to a unique combination of reduced numbers of osteoclasts that are non-functional. Fusion of mutant monocytes is deregulated and occurs rapidly and continuously to form giant, non-functional osteoclasts. Mutant osteoclasts mature quickly and survive poorly in vitro, possibly accounting for their scarcity in vivo. These cells also exhibit impaired ruffled borders, which are required for bone resorption, providing an additional basis for the osteopetrotic phenotype. More broadly, we propose that the maximal size of osteoclasts is actively determined by a genetically-regulated, cell-autonomous mechanism that limits prec...
Journal of Biological Chemistry, 2019
Bone morphogenetic proteins (BMPs) are important mediators of osteoclast differentiation. Although accumulating evidence has implicated BMPs in osteoblastogenesis, the mechanisms by which BMPs regulate osteoclastogenesis remain unclear. Activin A receptor type 1 (ACVR1) is a BMP type 1 receptor essential for skeletal development. Here, we observed that BMP-7, which preferentially binds to ACVR1, promotes osteoclast differentiation, suggesting ACVR1 is involved in osteoclastogenesis. To further investigate this, we isolated osteoclasts from either Acvr1-floxed mice or mice with constitutively activated Acvr1 (caAcvr1) carrying tamoxifeninducible Cre driven by a Ubiquitin promotor, and induced Cre activity in culture. Osteoclasts from the Acvr1-floxed mice had reduced osteoclast numbers and demineralization activity, whereas those from the caAcvr1-mutant mice formed large osteoclasts and demineralized pits, suggesting that BMP signaling through ACVR1 regulates osteoclast fusion and activity. It is reported that BMP-2 bind to BMPR1A, another BMP type 1 receptor, whereas BMP-7 bind to ACVR1 to activate SMAD1/5/9 signaling. Here, Bmpr1a-disrupted osteoclasts displayed reduced phospho-SMAD1/5/9 (pSMAD1/5/9) levels when induced by BMP-2, while no impacts on pSMAD1/5/9 were observed when induced by BMP-7. In contract, Acvr1-disrupted osteoclasts displayed reduced pSMAD1/5/9 levels when induced either by BMP-2 or BMP-7, suggesting that ACVR1 is the major receptor for transducing BMP-7 signals in osteoclasts. Indeed, LDN-193189 and LDN-212854-which specifically block SMAD1/5/9 phosphorylation-inhibited osteoclastogenesis of caAcvr1-mutant osteoclasts. Moreover, increased BMP signaling promoted nuclear translocation of nuclear factor of activated T cells 1 (NFATc1), which was inhibited by LDN treatments. Taken together, ACVR1-mediated BMP-SMAD signaling activates NFATc1, a regulatory protein crucial for RANKL-induced osteoclastogenesis. Osteoclasts are bone cells responsible for removing old bone and inducing osteoblastmediated bone formation, playing an essential role in renewing bone tissue and maintaining bone mass. However, pathological activation of osteoclast activity causes serious health conditions such as postmenopausal osteoporosis, Paget's disease, and bone cancer. Therefore, the mechanisms regulating osteoclast differentiation, including cell fusion and bone resorptive function, is critical for maintaining normal bone homeostasis. Osteoblasts secrete Macrophage Colony Stimulating Factor (M-CSF) and Receptor Activator of Nuclear factor-κB Ligand (RANKL) to regulate the activation of osteoclasts, and those factors are required for survival and differentiation of osteoclasts (1-3).
Decreased C-Src Expression Enhances Osteoblast Differentiation and Bone Formation
Journal of Cell Biology, 2000
c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src Ϫ / Ϫ relative to Src ϩ / ϩ mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by ف 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src Ϫ / Ϫ osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/ PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 2015
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 Rac1(flox/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. BMD was increased 21-33% in 4- to 6-week old DKO mice at all sites when measured by DXA and serum 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....
Deubiquitinating enzyme CYLD negatively regulates RANK signaling and osteoclastogenesis in mice
Journal of Clinical Investigation, 2008
Osteoclastogenesis is a tightly regulated biological process, and deregulation can lead to severe bone disorders such as osteoporosis. The regulation of osteoclastic signaling is incompletely understood, but ubiquitination of TNF receptor-associated factor 6 (TRAF6) has recently been shown to be important in mediating this process. We therefore investigated the role of the recently identified deubiquitinating enzyme CYLD in osteoclastogenesis and found that mice with a genetic deficiency of CYLD had aberrant osteoclast differentiation and developed severe osteoporosis. Cultured osteoclast precursors derived from CYLD-deficient mice were hyperresponsive to RANKL-induced differentiation and produced more and larger osteoclasts than did controls upon stimulation. We assessed the expression pattern of CYLD and found that it was drastically upregulated during RANKL-induced differentiation of preosteoclasts. Furthermore, CYLD negatively regulated RANK signaling by inhibiting TRAF6 ubiquitination and activation of downstream signaling events. Interestingly, we found that CYLD interacted physically with the signaling adaptor p62 and thereby was recruited to TRAF6. These findings establish CYLD as a crucial negative regulator of osteoclastogenesis and suggest its involvement in the p62/TRAF6 signaling axis.
Filamin A Regulates Monocyte Migration Through Rho Small GTPases During Osteoclastogenesis
Journal of Bone and Mineral Research, 2009
Osteoclastogenesis (OCG) results from the fusion of monocytes after stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Migration of monocytes into close proximity precedes critical fusion events that are required for osteoclast formation. Cellular migration requires leading-edge actin cytoskeleton assembly that drives cellular locomotion. Filamin A (FLNa) cross-links F-actin filaments in the leading edge of migrating cells and also has been shown to regulate signal transduction during cell migration. However, little is known about the possible role of FLNa in osteoclastogenesis. Our objective in this study was to investigate the role of FLNa in osteoclastogenesis. Bone marrow monocytes isolated from the tibiae and femora of wild type (WT) and Flna-null mice were cultured for 6 days with M-CSF and RANKL, and osteoclasts were identified by tartrate-resistant acid phosphatase (TRACP) staining. The Flna-null mouse skeletal phenotype was characterized using dual-energy X-ray absorptiometry (DXA) to analyze the skeleton, as well as tests on blood chemistry. Osteoclast levels in vivo were quantified by counting of TRACP-stained histologic sections of distal femora. To elucidate the mechanisms by which Flna regulates osteoclastogenesis, migration, actin polymerization, and activation of Rho GTPases, Rac1, Cdc42, and RhoA were assessed in monocytes during in vitro OCG. Deficiencies in migration were rescued using constitutively active Rac1 and Cdc42 TAT fusion proteins. The RANKL signaling pathway was evaluated for activation by monitoring nuclear translocation of NF kappaB and c-jun and expression of key osteoclast genes using quantitative real-time polymerase chain reaction (qRT-PCR). Our results show that Flna-null monocytes formed fewer osteoclasts in vitro, and those that were formed were smaller with fewer nuclei. Decreased OCG was reflected in vivo in TRACP-stained histologic bone sections. Flna-null monocytes experienced impaired migratory ability. When OCG was performed at increasing starting cellular plating densities in order to decrease intercellular distances, there was progressive rescue of Flna-null osteoclast formation comparable with WT levels, confirming that Flna regulates monocyte migration prefusion. Activation of the actin cytoskeleton regulators Rac1, Cdc42, and RhoA and actin free-barbed end generation were partially or completely abrogated in Flna-null monocytes; however, monocyte migration was restored on rescuing with constitutively active Rac1 and Cdc42 TAT fusion proteins. We conclude that filamin A is required for osteoclastogenesis by regulating actin dynamics via Rho GTPases that control monocyte migration.