Osteoclast-secreted CTHRC1 in the coupling of bone resorption to formation.
ABSTRACT: Bone remodeling is characterized by the sequential, local tethering of osteoclasts and osteoblasts and is key to the maintenance of bone integrity. While bone matrix-mobilized growth factors, such as TGF-β, are proposed to regulate remodeling, no in vivo evidence exists that an osteoclast-produced molecule serves as a coupling factor for bone resorption to formation. We found that CTHRC1, a protein secreted by mature bone-resorbing osteoclasts, targets stromal cells to stimulate osteogenesis. Cthrc1 expression was robustly induced when mature osteoclasts were placed on dentin or hydroxyapatite, and also by increasing extracellular calcium. Cthrc1 expression in bone increased in a high-turnover state (such as that induced by RANKL injections in vivo), but decreased in conditions associated with suppressed bone turnover (such as with aging and after alendronate treatment). Targeted deletion of Cthrc1 in mice eliminated Cthrc1 expression in bone, whereas its deficiency in osteoblasts did not exert any significant effect. Osteoclast-specific deletion of Cthrc1 resulted in osteopenia due to reduced bone formation and impaired the coupling process after resorption induced by RANKL injections, impairing bone mass recovery. These data demonstrate that CTHRC1 is an osteoclast-secreted coupling factor that regulates bone remodeling.
Project description:Bone remodeling is characterized by the sequential, local tethering of osteoclasts and osteoblasts, and is key to the maintenance of bone integrity. While bone matrix-mobilized growth factors, such as TGF-β, are proposed to regulate remodeling, no in vivo evidence exists that an osteoclast-produced molecule is the enigmatic coupling factor. We have identified Cthrc1, a protein secreted by mature bone-resorbing osteoclasts, that targets stromal cells so as to stimulate osteogenesis. The expression of Cthrc1 is robustly induced when mature osteoclasts are placed on dentin or hydroxyapatite, and also by increasing extracellular calcium. Cthrc1 expression in bone increases in a high turnover state, such as that which is induced by RANKL injections in vivo, whereas it decreases with aging or following alendronate treatment, conditions associated with suppressed bone turnover. The targeted deletion of the Cthrc1 gene eliminates Cthrc1 expression in bone, whereas its deficiency in osteoblasts does not exert any significant effect. Osteoclast-specific deletion of the Cthrc1 gene results in osteopenia due to reduced bone formation: it also impairs the coupling process following resorption induced by RANKL injections, with a resultant impairment of bone mass recovery. Thus, Cthrc1 is an osteoclast-secreted “coupling factor” that regulates bone remodeling and hence, skeletal integrity. Total bone marrow cells were prepared from the femurs and tibias of 8-10-week-old C57BL/6 mice and cultured in the presence of M-CSF (100ng/ml) for 3 days as described previously (Takeshita et al., 2000 JBMR 15:1477-1488). Cells were harvested with 0.02% EDTA/PBS and used as bone marrow macrophages (BMMs). These BMMs were cultured in the presence of M-CSF (100 ng/ml) and RANKL (100ng/ml) for 2 days. TRAP positive mononuclear cells were harvested and used as pre-osteoclasts (pOC). These pOC cells were further cultured in the presence of M-CSF and RANKL for 2 days in normal plastic plate or on dentin slices. After 2 days, multinucleated TRAP positive mature osteoclasts were generated as mature osteoclasts on plate (mOCp) and mature resorbing osteoclasts on dentin (mOCd), respectively. RNAs were extracted from four different stages of osteoclast lineage cells; BMMs, pOC, mOCp and mOCd, and used for microarray analysis.
Project description:Collagen triple helix repeat-containing1 (Cthrc1) has previously been implicated in osteogenic differentiation and positive regulation of bone mass, however, the underlying mechanisms remain unclear. Here we characterized the bone phenotype of a novel Cthrc1 null mouse strain using bone histomorphometry, ?CT analysis and functional readouts for bone strength. In male Cthrc1 null mice both trabecular bone as well as cortical bone formation was impaired, whereas in female Cthrc1 null mice only trabecular bone parameters were altered. Novel and highly specific monoclonal antibodies revealed that CTHRC1 is expressed by osteocytes and osteoblasts, but not osteoclasts. Furthermore, Cthrc1 null mice exhibited increased bone resorption with increased number of osteoclast and increased osteoclast activity together with enhanced expression of osteoclastogenic genes such as c-Fos, Rankl, Trap, and Nfatc1. Differentiation of bone marrow-derived monocytes isolated from Cthrc1 null mice differentiated into osteoclasts as effectively as those from wildtype mice. In the presence of CTHRC1 osteoclastogenic differentiation of bone marrow-derived monocytes was dramatically inhibited as was functional bone resorption by osteoclasts. This process was accompanied by downregulation of osteoclastogenic marker genes, indicating that extrinsically derived CTHRC1 is required for such activity. In vitro, CTHRC1 had no effect on osteogenic differentiation of bone marrow stromal cells, however, calvarial osteoblasts from Cthrc1 null mice exhibited reduced osteogenic differentiation compared to osteoblasts from wildtypes. In a collagen antibody-induced arthritis model Cthrc1 null mice suffered significantly more severe inflammation and joint destruction than wildtypes, suggesting that CTHRC1 expressed by the activated synoviocytes has anti-inflammatory effects. Mechanistically, we found that CTHRC1 inhibited NF?B activation by preventing I?B? degradation while also inhibiting ERK1/2 activation. Collectively our studies demonstrate that CTHRC1 secreted from osteocytes and osteoblasts functions as an inhibitor of osteoclast differentiation via inhibition of NF?B-dependent signaling. Furthermore, our data suggest that CTHRC1 has potent anti-inflammatory properties that limit arthritic joint destruction.
Project description:Physiological bone remodeling requires that bone formation by osteoblasts be tightly coupled to bone resorption by osteoclasts. However, relatively little is understood about how this coupling is regulated. Here, we demonstrate that modulation of NF-?B signaling in osteoclasts via a novel activity of charged multivesicular body protein 5 (CHMP5) is a key determinant of systemic rates of bone turnover. A conditional deletion of CHMP5 in osteoclasts leads to increased bone resorption by osteoclasts coupled with exuberant bone formation by osteoblasts, resembling an early onset, polyostotic form of human Paget's disease of bone (PDB). These phenotypes are reversed by haploinsufficiency for Rank, as well as by antiresorptive treatments, including alendronate, zolendronate, and OPG-Fc. Accordingly, CHMP5-deficient osteoclasts display increased RANKL-induced NF-?B activation and osteoclast differentiation. Biochemical analysis demonstrated that CHMP5 cooperates with the PDB genetic risk factor valosin-containing protein (VCP/p97) to stabilize the inhibitor of NF-?B? (I?B?), down-regulating ubiquitination of I?B? via the deubiquitinating enzyme USP15. Thus, CHMP5 tunes NF-?B signaling downstream of RANK in osteoclasts to dampen osteoclast differentiation, osteoblast coupling and bone turnover rates, and disruption of CHMP5 activity results in a PDB-like skeletal disorder.
Project description:Osteoclasts resorb the mineralized matrices formed by chondrocytes or osteoblasts. The cytokine receptor activator of nuclear factor-?B ligand (RANKL) is essential for osteoclast formation and thought to be supplied by osteoblasts or their precursors, thereby linking bone formation to resorption. However, RANKL is expressed by a variety of cell types, and it is unclear which of them are essential sources for osteoclast formation. Here we have used a mouse strain in which RANKL can be conditionally deleted and a series of Cre-deleter strains to demonstrate that hypertrophic chondrocytes and osteocytes, both of which are embedded in matrix, are essential sources of the RANKL that controls mineralized cartilage resorption and bone remodeling, respectively. Moreover, osteocyte RANKL is responsible for the bone loss associated with unloading. Contrary to the current paradigm, RANKL produced by osteoblasts or their progenitors does not contribute to adult bone remodeling. These results suggest that the rate-limiting step of matrix resorption is controlled by cells embedded within the matrix itself.
Project description:Bone remodeling is a continuous process to retain the structural integrity and function of the skeleton. A tight coupling is maintained between osteoclast-mediated resorption of old or damaged bones and osteoblast-mediated formation of new bones for bone homeostasis. While osteoblasts differentiate from mesenchymal stem cells, osteoclasts are hematopoietic in origin and derived from myeloid precursor cells. Osteoclast differentiation is driven by two cytokines, cytokine receptor activator of NF-?B ligand (RANKL), and macrophage colony-stimulating factor. Imbalances in the activity of osteoblasts and osteoclasts result in the development of bone disorders. Bacterially caused porcine atrophic rhinitis is characterized by a loss of nasal ventral conche bones and a distortion of the snout. While <i>Bordetella bronchiseptica</i> strains cause mild and reversible symptoms, infection of pigs with toxigenic <i>Pasteurella multocida</i> strains causes a severe and irreversible decay. The responsible virulence factor <i>Pasteurella multocida</i> toxin (PMT) contains a deamidase activity in its catalytical domain that constitutively activates specific heterotrimeric G proteins to induce downstream signaling cascades. While osteoblasts are inhibited by the toxin, osteoclasts are activated, thus skewing bone remodeling toward excessive bone degradation. Still, the mechanism by which PMT interferes with bone homeostasis, and the reason for this unusual target tissue is not yet well understood. Here, we show that PMT has the potential to differentiate bone marrow-derived macrophages into functional osteoclasts. This toxin-mediated differentiation process is independent of RANKL, a cytokine believed to be indispensable for triggering osteoclastogenesis, as addition of osteoprotegerin to PMT-treated macrophages does not show any effect on PMT-induced osteoclast formation. Although RANKL is not a prerequisite, toxin-primed macrophages show enhanced responsiveness to low concentrations of RANKL, suggesting that the PMT-generated microenvironment offers conditions where low concentrations of RANKL lead to an increase in the number of osteoclasts resulting in increased resorption. PMT-mediated release of the osteoclastogenic cytokines such as IL-6 and TNF-?, but not IL-1, supports the differentiation process. Although the production of cytokines and the subsequent activation of signaling cascades are necessary for PMT-mediated differentiation into osteoclasts, they are not sufficient and PMT-induced activation of G protein signaling is essential for efficient osteoclastogenesis.
Project description:Honeybush tea, a sweet tasting caffeine-free tea that is indigenous to South Africa, is rich in bioactive compounds that may have beneficial health effects. Bone remodeling is a physiological process that involves the synthesis of bone matrix by osteoblasts and resorption of bone by osteoclasts. When resorption exceeds formation, bone remodeling can be disrupted resulting in bone diseases such as osteoporosis. Osteoclasts are multinucleated cells derived from hematopoietic precursors of monocytic lineage. These precursors fuse and differentiate into mature osteoclasts in the presence of receptor activator of NF-kB ligand (RANKL), produced by osteoblasts. In this study, the in vitro effects of an aqueous extract of fermented honeybush tea were examined on osteoclast formation and bone resorption in RAW264.7 murine macrophages. We found that commercial honeybush tea extract inhibited osteoclast formation and TRAP activity which was accompanied by reduced bone resorption and disruption of characteristic cytoskeletal elements of mature osteoclasts without cytotoxicity. Furthermore, honeybush tea extract decreased expression of key osteoclast specific genes, matrix metalloproteinase-9 (MMP-9), tartrate resistant acid phosphatase (TRAP) and cathepsin K. This study demonstrates for the first time that honeybush tea may have potential anti-osteoclastogenic effects and therefore should be further explored for its beneficial effects on bone.
Project description:Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.
Project description:Bone remodeling, a physiological process characterized by bone formation by osteoblasts and bone resorption by osteoclasts, is important for the maintenance of healthy bone in adult humans. Osteoclasts play a critical role in bone erosion and osteoporosis and are bone-specific multinucleated cells generated through differentiation of monocyte/macrophage lineage precursors. Receptor activator of NF-?B ligand (RANKL) has been reported to induce osteoclast differentiation. In this study, we explored whether Gracilaria verrucosa extracts (GE) could affect RANKL-mediated osteoclast differentiation. GE significantly inhibited RANKL-activated osteoclast differentiation by inhibiting protein expression of c-fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), vital factors in RANKL-mediated osteoclastogenesis. In addition, GE attenuated ovariectomy-induced bone loss in mice. In summary, GE can prevent osteoclastogenesis and hormone-related bone loss via blockage of c-fos-NFATc1 signaling. Our results suggest that GE may have therapeutic potential in the treatment of postmenopausal osteoporosis.
Project description:Osteoporosis is a metabolic bone disease with dysregulated coupling between bone resorption and bone formation, which results in decreased bone mineral density. The MEF2C locus, which encodes the transcription factor MADS box transcription enhancer factor 2, polypeptide C (MEF2C), is strongly associated with adult osteoporosis and osteoporotic fractures. Although the role of MEF2C in bone and cartilage formation by osteoblasts, osteocytes, and chondrocytes has been studied, the role of MEF2C in osteoclasts, which mediate bone resorption, remains unclear. In this study, we identified MEF2C as a positive regulator of human and mouse osteoclast differentiation. While decreased MEF2C expression resulted in diminished osteoclastogenesis, ectopic expression of MEF2C enhanced osteoclast generation. Using transcriptomic and bioinformatic approaches, we found that MEF2C promotes the RANKL-mediated induction of the transcription factors c-FOS and NFATc1, which play a key role in osteoclastogenesis. Mechanistically, MEF2C binds to FOS regulatory regions to induce c-FOS expression, leading to the activation of NFATC1 and downstream osteoclastogenesis. Inducible deletion of Mef2c in mice resulted in increased bone mass under physiological conditions and protected mice from bone erosion by diminishing osteoclast formation in K/BxN serum induced arthritis, a murine model of inflammatory arthritis. Our findings reveal direct regulation of osteoclasts by MEF2C, thus adding osteoclasts as a cell type in which altered MEF2C expression or function can contribute to pathological bone remodeling.
Project description:Pathological bone loss is caused by an imbalance between bone formation and resorption. The bone microenvironments are hypoxic, and hypoxia-inducible factor (HIF) is known to play notable roles in bone remodeling. However, the relevant functions of HIF-2α are not well understood. Here, we have shown that HIF-2α deficiency in mice enhances bone mass through its effects on the differentiation of osteoblasts and osteoclasts. In vitro analyses revealed that HIF-2α inhibits osteoblast differentiation by targeting Twist2 and stimulates RANKL-induced osteoclastogenesis via regulation of Traf6. In addition, HIF-2α appears to contribute to the crosstalk between osteoblasts and osteoclasts by directly targeting RANKL in osteoprogenitor cells. Experiments performed with osteoblast- and osteoclast-specific conditional knockout mice supported a role of HIF-2α in this crosstalk. HIF-2α deficiency alleviated ovariectomy-induced bone loss in mice, and specific inhibition of HIF-2α with ZINC04179524 significantly blocked RANKL-mediated osteoclastogenesis. Collectively, our results suggest that HIF-2α functions as a catabolic regulator in bone remodeling, which is critical for the maintenance of bone homeostasis.