Modulator Of Osteoclast Differentiation Research Articles

Chalcone T4 Inhibits RANKL-Induced Osteoclastogenesis and Stimulates Osteogenesis In Vitro.

Chalcones are phenolic compounds produced during the biosynthesis of flavonoids that have numerous biological activities, including anti-inflammatory, antioxidant and anticancer. In this in vitro study, we investigate a newly synthesized chalcone (Chalcone T4) in the context of bone turnover, specifically on the modulation of osteoclast differentiation and activity and osteoblast differentiation. Murine macrophages (RAW 264.7) and pre-osteoblasts (MC3T3-E1) were used as models of osteoclasts and osteoblasts, respectively. Differentiation and activity osteoclasts were induced by RANKL in the presence and absence of non-cytotoxic concentrations of Chalcone T4, added in different periods during osteoclastogenesis. Osteoclast differentiation and activity were assessed by actin ring formation and resorption pit assay, respectively. Expression of osteoclast-specific markers (Nfatc1, Oscar, Acp5, Mmp-9 and Ctsk) was determined by RT-qPCR, and the activation status of relevant intracellular signaling pathways (MAPK, AKT and NF-kB) by Western blot. Osteoblast differentiation and activity was induced by osteogenic culture medium in the presence and absence of the same concentrations of Chalcone T4. Outcomes assessed were the formation of mineralization nodules via alizarin red staining and the expression of osteoblast-related genes (Alp e Runx2) by RT-qPCR. Chalcone T4 reduced RANKL-induced osteoclast differentiation and activity, suppressed Oscar, Acp5 and Mmp-9 expression, and decreased ERK and AKT activation in a dose-dependent manner. Nfact1 expression and NF-kB phosphorylation were not modulated by the compound. Mineralized matrix formation and the expression of Alp and Runx2 by MC3T3-E1 cells were markedly stimulated by Chalcone T4. Collectively, these results demonstrate that Chalcone T4 inhibits in osteoclast differentiation and activity and stimulates osteogenesis, which indicates a promising therapeutic potential in osteolytic diseases.

Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1

Activated osteoclasts release large amounts of small extracellular vesicles (sEVs) during bone remodeling. However, little is known about whether osteoclast-derived sEVs affect surrounding cells. In this study, osteoclasts were generated by stimulating bone marrow macrophages (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear actor κB ligand (RANKL). We performed microarray analysis of sEV-microRNAs (miRNAs)s secreted from osteoclast at different stages and identified four miRNAs that were highly expressed in mature osteoclast-derived sEVs. One of these miRNAs, miR-324, significantly induced osteogenic differentiation and mineralization of primary mesenchymal stem cells (MSCs) in vitro by targeting ARHGAP1, a negative regulator of osteogenic differentiation. We next fabricated an sEV-modified scaffold by coating decalcified bone matrix (DBM) with osteoclast-derived sEVs, and the pro-osteogenic regeneration activities of the sEV-modified scaffold were validated in a mouse calvarial defect model. Notably, miR-324-enriched sEV-modified scaffold showed the highest capacity on bone regeneration, whereas inhibition of miR-324 in sEVs abrogated these effects. Taken together, our findings suggest that miR-324-contained sEVs released from mature osteoclast play an essential role in the regulation of osteogenic differentiation and potentially bridge the coupling between osteoclasts and MSCs.

Noncanonical G-protein-dependent modulation of osteoclast differentiation and bone resorption mediated by Pasteurella multocida toxin.

ABSTRACTPasteurella multocida toxin (PMT) induces atrophic rhinitis in animals, which is characterized by a degradation of nasal turbinate bones, indicating an effect of the toxin on bone cells such as osteoblasts and osteoclasts. The underlying molecular mechanism of PMT was defined as a persistent activation of heterotrimeric G proteins by deamidation of a specific glutamine residue. Here, we show that PMT acts directly on osteoclast precursor cells such as bone marrow-derived CD14+ monocytes and RAW246.7 cells to induce osteoclastogenesis as measured by expression of osteoclast-specific markers such as tartrate-resistant acid phosphatase and bone resorption activity. Treatment performed solely with PMT stimulates osteoclast differentiation, showing a receptor activator of nuclear factor-κB ligand (RANKL)-independent action of the toxin. The underlying signal transduction pathway was defined as activation of the heterotrimeric G proteins Gαq/11 leading to the transactivation of Ras and the mitogen-activated protein kinase pathway. Gαq/11 transactivates Ras via its effector phospholipase Cβ-protein kinase C (PKC) involving proline-rich tyrosine kinase 2 (Pyk2). PMT-induced activation of the mitogen-activated protein kinase pathway results in stimulation of the osteoclastogenic transcription factors AP-1, NF-κB, and NFATc1. In addition, Ca2+-dependent calcineurin activation of NFAT is crucial for PMT-induced osteoclastogenesis. The data not only elucidate a rationale for PMT-dependent bone loss during atrophic rhinitis but also highlight a noncanonical, G-protein-dependent pathway toward bone resorption that is distinct from the RANKL-RANK pathway but mimics it. We define heterotrimeric G proteins as as-yet-underestimated entities/players in the maturation of osteoclasts which might be of pharmacological relevance.

Inpp4b is a novel negative modulator of osteoclast differentiation and a prognostic locus for human osteoporosis

Inositol polyphosphate 4-phosphatase type II (Inpp4b) is a novel negative modulator of osteoclast differentiation and a prognostic locus for human osteoporosis. This short overview summarizes some of the cellular, molecular, and crosstalk signaling mechanisms that control osteoclast and osteoblast differentiation and activation.

Hydroxyapatite surface roughness: Complex modulation of the osteoclastogenesis of human precursor cells

It is recognized that the surface roughness affects osteoblastic differentiation, but little information is available regarding its effect on osteoclastogenesis. With this work, the osteoclastogenic behaviour of human peripheral blood mononuclear cells (PBMCs), cultured isolated (1.5×106cellscm−2) or co-cultured with human bone marrow cells (hBMCs; 103cellscm−2), was assessed on surface-abraded hydroxyapatite disks with three different surface roughnesses (Ra 0.0437–0.582μm). Monocultures and co-cultures were performed for 21days in the absence or presence of recombinant M-CSF and RANKL. Results showed that PBMCs supplemented with M-CSF and RANKL or co-cultured with hBMCs displayed typical osteoclastic features, i.e. multinucleated cells with actin rings, vitronectin and calcitonin receptors, gene expression of TRAP, cathepsin K, carbonic anhydrase 2, c-myc and c-src, TRAP activity and resorbing activity. The osteoclastogenic response increased with surface roughness in PBMCs cultured with M-CSF and RANKL but decreased in PBMCs co-cultured with hBMCs. However, co-cultures supplemented with the osteoclastogenic inducers displayed high and similar levels of osteoclast differentiation in the three tested surfaces. In conclusion, modulation of osteoclast differentiation by surface roughness seemed to be dependent on the mechanisms subjacent to the osteoclastogenic stimulus, i.e. the presence of soluble factors or direct cell-to-cell contacts between osteoblastic and osteoclastic cells.

A new role for OPG: Putting RANKL in its place

The receptor activator of NF-kB (RANK), its cognate ligand (RANKL), and the secreted decoy receptor osteoprotegrin (OPG) are critical molecules that regulate bone remodeling through modulation of osteoclast differentiation and function. (1) The former is expressed on the surface of the myeloid precursors of osteoclasts as well as on mature osteoclasts, whereas the latter two are expressed by osteoblasts. Genetic evidence obtained from both mouse knockout models and human studies demonstrate that the genes encoding these proteins are required for normal bone modeling and remodeling. OPG forms a complex with RANKL that prevents the RANKL-RANK interaction necessary for downstream signal activation in osteoclast precursors. It is generally accepted that this system is balanced by the ratio of RANKL to secreted OPG. This ratio is believed to be critical for maintaining bone homeostasis and is disrupted in bone disease states, for example, in postmenopausalosteoporosis and Pagetdisease. However, thedogma that the balance between membrane-bound RANKL and secreted OPG decoy receptor as the mechanism underlying control of osteoclast differentiation and function is challenged by the elegant work of Masashi Honma, Hiroshi Suzuki, and colleagues presented in the current issue. (2) This work builds on previous studies by this same group demonstrating that the majority of newly synthesized RANKL in osteoblasts is not transported to the cell surface but shuttled from the Golgi apparatus to secretory lysosomes, where it is stored. (3) A small portion of RANKL is transported directly from the Golgi apparatus to the cell surface by what is referred by these authors to as the ‘‘alternative pathway.’’ When this cell surface RANKL makes contact with beads coated with RANK, the RANKLinthelysosomalpooltravelstothecellsurface. (3) Thenew work starts with the premise that OPG may be involved in RANKL transport to the secretory lysosome. Cell biologic approaches are used to demonstrate that in OPG / osteoblasts RANKL accumulates in the Golgi apparatus, but reintroduction of OPG results in accumulation in the lysosomal compartment. This type of assay was used to demonstrate that the three known domains of OPG, the N-terminal cysteine-rich domain (CRD) that mediates binding to RANKL, the death domain homologous domain, and the C-terminal heparin-binding domain are all required and act in concert to promote lysosomal trafficking of RANKL. The mechanism of OPG action seems to involve enhancing the interaction of RANKL with components of the machinery for lysosomal transport such as Vps33a. The most convincing evidence for the biologic significance of RANKL lysosomal trafficking comes from experiments with a chimeric OPG protein in which the CRD of RANK is substituted for the OPG CRD domain. In a coculture assay with the stromal ST2 cell line, addition of the chimeric RANK-CRD-OPG protein to the cell medium inhibits the osteoclastogenic activity of the ST2 cells; that is, the chimeric protein acts as an effective decoy receptor. In contrast, when coexpressed in ST2 cells with green fluorescent protein (GFP)-RANKL, the RANK-CRD-OPG protein

Growth/differentiation factor-15 inhibits differentiation into osteoclasts—A novel factor involved in control of osteoclast differentiation

Survival and capability of cancer cells to form metastases fundamentally depend on interactions with their microenvironment. Secondary tumors originating from prostate carcinomas affect remodeling of bone tissue and can induce both osteolytic and osteocondensing lesions. However, particular molecular mechanisms responsible for selective homing and activity of cancer cells in bone microenvironment have not been clarified yet. Growth/differentiation factor-15 (GDF-15), a distant member of the TGF-β protein family, has recently been associated with many human cancers, including prostate. We show that both pure GDF-15 and the GDF-15-containing growth medium of 1,25(OH) 2-vitamin D 3-treated prostate adenocarcinoma LNCaP cells suppress formation of mature osteoclasts differentiated from RAW264.7 macrophages and bone-marrow precursors by M-CSF/RANKL in a dose-dependent manner. GDF-15 inhibits expression of c-Fos and activity of NFκB by delayed degradation of IκB. Moreover, GDF-15 inhibits expression of carbonic anhydrase II and cathepsin K, key osteoclast enzymes, and induces changes in SMAD and p38 signaling. The lack of functional osteoclasts can contribute to accumulation of bone matrix by reduction of bone resorption. These results unveil new role of GDF-15 in modulation of osteoclast differentiation and possibly in therapy of bone metastases.

Lysyl Oxidase Binds Transforming Growth Factor-; and Regulates Its Signaling via Amine Oxidase Activity

Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-beta1 (TGF-beta1), a potent growth factor abundant in bone, the effect of LOX on TGF-beta1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-beta1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-beta1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-beta1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling.

Variant of Adynamic Bone Disease in Hemodialysis Patients: Fact or Fiction?

Adynamic bone disease is a type of renal osteodystrophy characterized by low bone turnover and paucity of bone cells. It was proposed that a new type of this disease featuring high osteoclastic resorption without parathyroid hormone stimulus and designated adynamic bone disease variant occurs in hemodialysis patients. The present study is designed to evaluate the frequency and characteristics of both diseases in a large series of bone biopsy specimens. We reviewed 1,160 bone biopsy specimens from hemodialysis patients. Specimens in which adynamic bone disease was diagnosed were selected and categorized as classic or variant based on osteoclastic surface. In 218 bone biopsy specimens (18.8%), adynamic bone disease was identified, whereas the variant form was identified in 35 specimens (38.8%). Biopsy specimens categorized as the variant form were from patients who were younger and had greater phosphorus and parathyroid hormone levels. Histologically, the variant form presented greater osteoid volume, fibrosis volume, osteoid surface, osteoblast surface, and eroded surface. Similarly, values for all dynamic parameters were greater in the variant group. Osteoclastic surface correlated with phosphorus level, parathyroid hormone level, and osteoblast surface. Age and osteoblast surface were identified as independent determinants of the variant form. Adynamic bone disease variant seems to occur in younger hemodialysis patients with greater levels of parathyroid hormone, which acts on cell-covered bone surfaces. It probably is a transitional phase from low- to high-turnover status, rather than a true entity within the spectrum of renal osteodystrophy.

RANKL-RANKシグナルによる破骨細胞の分化調節 : RANKLの発見から骨免疫学の展開まで

破骨細胞は骨吸収を司る多核巨細胞であり,骨吸収因子によって骨芽細胞/骨髄ストローマ細胞の細胞膜表面に誘導されるRANKLと破骨細胞前駆細胞が発現するRANKとの細胞間接触を介して誘導される.さらに遺伝子ターゲティングの結果から,RANKL, RANKが破骨細胞分化に必須の因子であることが証明された.以前から,局所に浸潤するT細胞やリンパ球やこれらの細胞から産生されるサイトカインなどが,骨破壊に関与することが知られていたが,近年,免疫系細胞による骨代謝調節機構を解明する研究として「骨免疫学」が提唱され,発展している.例えば,RANKLは,カルシウム依存性のカルシニューリンを介して特異的にかつ持続的にT細胞の活性化に重要な役割を担う転写因子,NFATc1を活性化する.NFATc1を欠損したES細胞から破骨細胞は誘導されず,またNFATc1遺伝子を破骨細胞前駆細胞に強制発現させるとRANKL刺激なしに破骨細胞を誘導できることから,NFATc1は破骨細胞のマスター分子と考えられる.骨代謝と免疫応答を結び付けるもうひとつの因子はNF-κBである.NF-κBのp50,p52二重欠損マウスは大理石骨病を呈し,IKKα,やNIK欠損マウスでも破骨細胞形成の抑制が認められる.さらにNF-κBの活性化経路を選択的に阻害するNBDペプチドは破骨細胞形成を抑制する.NF-κBが破骨細胞分化に必須の因子であることは広く受け入れられているが,NF-κBの標的遺伝子を同定し,NF-κBによる破骨細胞分化の分子メカニズムを解明することが重要である.

Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families.

Osteoblasts/stromal cells are essentially involved in osteoclast differentiation and function through cell-to-cell contact (Fig. 8). Although many attempts have been made to elucidate the mechanism of the so-called "microenvironment provided by osteoblasts/stromal cells," (5-8) it has remained an open question until OPG and its binding molecule were cloned. The serial discovery of the new members of the TNF receptor-ligand family members has confirmed the idea that osteoclast differentiation and function are regulated by osteoblasts/stromal cells. RANKL, which has also been called ODF, TRANCE, or OPGL, is a member of the TNF ligand family. Expression of RANKL mRNA in osteoblasts/stromal cells is up-regulated by osteotropic factors such as 1 alpha, 25(OH)2D3, PTH, and IL-11. Osteoclast precursors express RANK, a TNF receptor family member, recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into pOCs in the presence of M-CSF. RANKL is also involved in the survival and fusion of pOCs and activation of mature osteoclasts. OPG, which has also been called OCIF or TR1, is a soluble receptor for RANKL and acts as a decoy receptor in the RANK-RANKL signaling system (Fig. 8). In conclusion, osteoblasts/stromal cells are involved in all of the processes of osteoclast development, such as differentiation, survival, fusion, and activation of osteoclasts (Fig. 8). Osteoblasts/stromal cells can now be replaced with RANKL and M-CSF in dealing with the whole life of osteoclasts. RANKL, RANK, and OPG are three key molecules that regulate osteoclast recruitment and function. Further studies on these key molecules will elucidate the molecular mechanism of the regulation of osteoclastic bone resorption. This line of studies will establish new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment and functions such as osteopetrosis, osteoporosis, metastatic bone disease, Paget's disease, rheumatoid arthritis, and periodontal bone disease.

Modulation of Osteoclast Differentiation and Function by the New Members of the Tumor Necrosis Factor Receptor and Ligand Families

Modulation of Osteoclast Differentiation and Function by the New Members of the Tumor Necrosis Factor Receptor and Ligand Families

Modulation of osteoclast differentiation by local factors.

Bone-resorbing osteoclasts are of hemopoietic cell origin, probably of the CFU-M-derived monocyte-macrophage family. Bone marrow-derived osteoblastic stromal cells play an important role in modulating the differentiation of osteoclast progenitors in two different ways: one is the production of soluble factors, and the other is cell-to-cell recognition between osteoclast progenitors and osteoblastic stromal cells. M-CSF is probably the most important soluble factor, which appears to be necessary for not only proliferation of osteoclast progenitors, but also differentiation into mature osteoclasts and their survival. A number of local factors as well as systemic hormones induce osteoclast differentiation. They are classified into three categories in terms of the signal transduction: vitamin D receptor-mediated signals [1 alpha,25(OH)2D3]; protein kinase A-mediated signals (PTH, PTHrP, PGE2, and IL-1); and gp130-mediated signals (IL-6, IL-11, oncostatin M, and leukemia inhibitory factor). All of these osteoclast-inducing factors appear to act on osteoblastic cells to commonly induce osteoclast differentiation factor (ODF), which recognizes osteoclast progenitors and prepares them to differentiate into mature osteoclasts. This line of approach will undoubtedly produce new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment such as osteoporosis, osteopetrosis, Paget's disease, rheumatoid arthritis, and periodontal disease.

Modulation of osteoclast differentiation [published erratum appears in Endocr Rev 1992 May;13(2):191

Modulation of osteoclast differentiation [published erratum appears in Endocr Rev 1992 May;13(2):191

Modulation of osteoclast differentiation.

I. Introduction BONE is a complex tissue in which resorption and formation continue throughout life. This process is called bone remodeling. Osteotropic hormones such as 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], PTH, and calcitonin preferentially modulate the process of bone resorption to maintain bone remodeling. The bone tissue contains various types of cells, of which the bone-forming osteoblasts and bone-resorbing osteoclasts are mainly responsible for bone remodeling. Osteoblasts are believed to be derived from undifferentiated mesenchymal cells, which further differentiate into osteocytes and are embedded in calcified tissues. Osteoclasts are multinucleated cells present only in bone. It is believed that osteoclast progenitors are of hemopoietic origin, and they are recruited from hemopoietic tissues such as bone marrow and circulating blood to bone. Osteoclast progenitors then proliferate and differentiate into mononuclear preosteoclasts and fuse with each other to form multinucleated osteoclasts. ...