Akiko Kukita

RANKL-induced DC-STAMP is essential for osteoclastogenesis.

Osteoclasts are bone-resorbing, multinucleated giant cells that are essential for bone remodeling and are formed through cell fusion of mononuclear precursor cells. Although receptor activator of nuclear factor–κB ligand (RANKL) has been demonstrated to be an important osteoclastogenic cytokine, the cell surface molecules involved in osteoclastogenesis are mostly unknown. Here, we report that the seven-transmembrane receptor-like molecule, dendritic cell–specific transmembrane protein (DC-STAMP) is involved in osteoclastogenesis. Expression of DC-STAMP is rapidly induced in osteoclast precursor cells by RANKL and other osteoclastogenic stimulations. Targeted inhibition of DC-STAMP by small interfering RNAs and specific antibody markedly suppressed the formation of multinucleated osteoclast-like cells. Overexpression of DC-STAMP enhanced osteoclastogenesis in the presence of RANKL. Furthermore, DC-STAMP directly induced the expression of the osteoclast marker tartrate-resistant acid phosphatase. These data demonstrate for the first time that DC-STAMP has an essential role in osteoclastogenesis.

Inhibition of histone deacetylase suppresses osteoclastogenesis and bone destruction by inducing IFN-β production

Osteoclasts are bone-resorptive multinucleated cells that are differentiated from hemopoietic cell lineages of monocyte/macrophages in the presence of receptor activator of NF-κB ligand (RANKL) and M-CSF. Downstream signaling molecules of the receptor of RANKL, RANK, modulate the differentiation and the activation of osteoclasts. We recently found that histone deacetylase inhibitors (HDIs), known as anticancer agents, selectively suppressed osteoclastogenesis in vitro. However, the molecular mechanism underlying inhibitory action of HDIs in osteoclastogenesis and the effect of HDIs on pathological bone destruction are still not remained to be elucidated. In this study, we show that a depsipeptide, FR901228, inhibited osteoclast differentiation by not only suppressing RANKL-induced nuclear translocation of NFATc1 but also increasing the mRNA level of IFN-β, an inhibitor of osteoclastogenesis. The inhibition of osteoclast formation by FR901228 was abrogated by the addition of IFN-β-neutralizing Ab. In addition, treatment of adjuvant-induced arthritis in rats revealed that FR901228 inhibited not only disease development in a prophylactic model but also bone destruction in a therapeutic model. Furthermore, immunostaining of the joints of therapeutically treated rats revealed significant production of IFN-β in synovial cells. Taken together, these data suggest that a HDI inhibits osteoclastogenesis and bone destruction by a novel action to induce the expression of osteoclast inhibitory protein, IFN-β.

Fibroblasts from the inner granulation tissue of the pseudocapsule in hips at revision arthroplasty induce osteoclast differentiation, as do stromal cells

Background: It has previously been shown that many osteoclast precursors are included in the granulation tissue within the pseudocapsule obtained at revision arthroplasty from hips with osteolysis. In vitro culture of only cells isolated from the granulation tissue has been previously shown to generate many mature osteoclasts. Objective: To investigate the presence or otherwise of supporting cells, similar to stromal cells, which differentiate osteoclasts within the granulation tissue. Methods: Cells isolated from the granulation tissue were cultured alone, and after four weeks fibroblast-like cells (granulation fibroblasts) remained. Rat non-adherent bone marrow cells (NA-BMCs) were co-cultured with the granulation fibroblasts with or without 1α,25(OH)2D3 (10−8 M) or heat treated ROS 17/2.8 cell conditioned medium (ht ROSCM), or both. Multinucleated cells (MNCs), which formed, were assessed by biochemical and functional characterisation of osteoclasts. Receptor activator of NFκB ligand (RANKL) was investigated by immunohistochemistry. Results: Co-culture of NA-BMCs and granulation fibroblasts caused the formation of tartrate resistant acid phosphatase (TRAP) positive MNCs, which had the calcitonin receptor (CTR), the Kat-1 antigen, which is specific to the surface of rat osteoclasts, and the ability to form pits in the presence of both 1α,25(OH)2D3 and ht ROSCM or in the presence of just ht ROSCM. RANKL was detected in fibroblast-like cells in the granulation tissue. Conclusion: These data suggest that granulation fibroblasts support osteoclast differentiation, as do osteoblasts/stromal cells, and may play a part in aseptic loosening.

Possible involvement of MIP-1α in the recruitment of osteoclast progenitors to the distal tibia in rats with adjuvant-induced arthritis

In the rat model of rheumatoid arthritis, a marked formation of osteoclasts is found in the distal tibia and the metatarsal bone. It was therefore postulated that osteoclast progenitors would be increased in the bone marrow cavities of rats with adjuvant-induced arthritis (AA rats). Bone marrow cells obtained from tibia of AA rats were cultured to form cells in the osteoclast lineage to access the number of osteoclast progenitors. Unexpectedly, only a suppressed level of osteoclast progenitors was detected in the diaphyseal bone marrow of tibia in AA rats. Distribution of osteoclast progenitors in the bone marrow cavity was examined, and it was shown that osteoclast progenitors accumulated in the distal tibia. Macrophage inflammatory protein (MIP)-1α, an osteoclastogenic CC chemokine, was expressed in ED-1-positive macrophages localizing in the distal tibia with marked bone destruction. Chemotaxis studies showed that MIP-1α expressed significant activity towards bone marrow cells. The suppressed level of osteoclastogenesis in bone marrow cells of AA rats was restored to a normal level by the addition of MIP-1α. It was suggested that MIP-1α is involved in the migration of osteoclast progenitors to the distal tibia as well as in osteoclastogenesis in AA rats. In these rats, in situ hybridization of the distal tibia with a high level of bone destruction showed significant expression of Receptor activator nuclear factor κB ligand (RANKL) messenger RNA in aggregates of multinucleated osteoclast-like cells present in the bone marrow cavity, a unique pathological feature for these rats. Migrated osteoclast progenitors are thought to be efficiently differentiated into osteoclasts in response to RANKL expressed by the aggregates of osteoclast-like cells under the influence of the MIP-1α. Such positive-feedback regulation of osteoclastogenesis could result in the highest recruitment of active osteoclasts in the area of marked bone destruction.

Mesenchymal stem cells markedly suppress inflammatory bone destruction in rats with adjuvant-induced arthritis

Mesenchymal stem cells (MSCs) have potential to differentiate into multiple cell lineages. Recently, it was shown that MSCs also have anti-inflammatory and immunomodulatory functions. In this report, we investigated the regulatory function of MSCs in the development of inflammatory bone destruction in rats with adjuvant-induced arthritis (AA rats). MSCs were isolated from rat bone marrow tissues, expanded in the presence of basic FGF, and intraperitoneally injected into AA rats. MSC administration significantly suppressed inflammatory parameters: swelling score, swelling width, and thickness of hind paw. Radiographic evaluation indicated that MSC significantly suppressed bone destruction. Histological analysis showed that administration of MSCs markedly suppressed osteoclastogenesis in AA rats. To further delineate their effects on osteoclastogenesis, MSCs were added to in vitro bone marrow cultures undergoing osteoclastogenesis. MSCs significantly suppressed osteoclastogenesis in this system. Chemokine receptor expression in MSCs was assessed by RT-PCR, and a chemotactic assay was performed using a transwell culture system. MSCs showed significant chemotaxis to MIP-1α (CCL3) and SDF-1α (CXCL12), chemokines preferentially expressed in the area of inflammatory bone destruction. Furthermore, MSCs expressed IL-10 and osteoprotegerin, cytokines that suppress osteoclastogenesis. These data suggest that recruitment of MSC to the area of bone destruction in AA rats could suppress inflammatory bone destruction and raise the possibility that MSCs may have potential for the treatment of inflammatory bone destruction in arthritis.

Tunneling nanotube formation is essential for the regulation of osteoclastogenesis

Osteoclasts are the multinucleated giant cells formed by cell fusion of mononuclear osteoclast precursors. Despite the finding of several membrane proteins involving DC‐STAMP as regulatory proteins required for fusion among osteoclast precursors, cellular and molecular events concerning this process are still ambiguous. Here we identified Tunneling Nanotubes (TNTs), long intercellular bridges with small diameters, as the essential cellular structure for intercellular communication among osteoclast precursors in prior to cell fusion. Formation of TNTs was highly associated with osteoclastogenesis and it was accompanied with the significant induction of the M‐Sec gene, an essential gene for TNT formation. M‐Sec gene expression was significantly upregulated by RANKL‐treatment in osteoclast precursor cell line. Blockage of TNT formation by Latrunclin B or by M‐Sec siRNA significantly suppressed osteoclastogenesis. We have detected the rapid intercellular transport of not only the membrane phospholipids labeled with DiI but also the DC‐STAMP‐GFP fusion protein through TNTs formed among osteoclast precursors during osteoclastogenesis. Transportation of such regulatory molecules through TNTs would be essential for the process of the specific cell fusion among osteoclast precursors. J. Cell. Biochem. 114: 1238–1247, 2013.

Mature and activated osteoclasts exist in the synovium of rapidly destructive coxarthrosis

We compared histological and functional findings in rapidly destructive coxarthrosis (RDC) and slowly progressive osteoarthritis (OA) to investigate whether osteoclasts contribute to the extensive bone destruction observed in RDC. A histological analysis of tissue specimens from the synovium obtained from 10 cases of RDC and 40 cases with OA of the hip was performed after staining for tartrate-resistant acid phosphatase (TRAP). The cells isolated from these tissue specimens from the synovium were cultured for 24 h, and the numbers of TRAP-positive giant cells were counted. Thereafter, we performed a resorption pit formation assay by isolated cells cultured on dentine slices for 7 days. The number of TRAP-positive multinuclear giant cells present in the synovial membrane obtained from RDC patients was significantly larger than that obtained from OA patients. Large lacunar resorption pits were only seen on the dentin slices in a culture of isolated cells from RDC patients without any stimulators. This is the first report, to our knowledge, to reveal that mature and activated osteoclasts exist only in the synovium of RDC and not in the OA synovium. This result might suggest that the underlying mechanism of RDC is therefore associated with osteoclastogenesis in the synovium.

Stage-specific functions of leukemia/lymphoma-related factor (LRF) in the transcriptional control of osteoclast development

Cell fate determination is tightly regulated by transcriptional activators and repressors. Leukemia/lymphoma-related factor (LRF; encoded by Zbtb7a), known as a POK (POZ/BTB and Krüppel) family transcriptional repressor, is induced during the development of bone-resorbing osteoclasts, but the physiological significance of LRF in bone metabolism and the molecular mechanisms underlying the transcriptional regulation of osteoclastogenesis by LRF have not been elucidated. Here we show that LRF negatively regulates osteoclast differentiation by repressing nuclear factor of activated T cells c1 (NFATc1) induction in the early phase of osteoclast development, while positively regulating osteoclast-specific genes by functioning as a coactivator of NFATc1 in the bone resorption phase. The stage-specific distinct functions of LRF were demonstrated in two lines of conditional knockout mice in which LRF was deleted in the early or late phase of osteoclast development. Thus, this study shows that LRF plays stage-specific distinct roles in osteoclast differentiation, exemplifying the delicate transcriptional regulation at work in lineage commitment.

The human apoptosis inhibitor NAIP induces pyroptosis in macrophages infected with Legionella pneumophila.

Human nucleotide oligomerization domain-like receptor family apoptosis inhibitory protein (NAIP) prevents apoptosis by inhibiting caspase-3, -7, and -9. Four functional Naip exist in the murine genome, each of which is equally similar to human NAIP. Among them, Naip5 induces pyroptosis by promoting caspase-1 activation in response to Legionella pneumophila infection in macrophages. However, the contribution of human NAIP to this response is unclear. To investigate the role of human NAIP in macrophage survival, we stably expressed human NAIP in RAW264.7 macrophages. Human NAIP inhibited camptothecin-induced apoptosis in macrophages; however, it promoted cytotoxicity in L. pneumophila-infected cells. This cytotoxicity was associated with caspase-1. In addition, human NAIP restricted the intracellular growth of L. pneumophila. L. pneumophila flagellin was required for cytotoxicity, caspase-1 activation, and restriction of intracellular bacterial growth. Expression of murine Naip5 produced comparable results. These data indicate that human NAIP regulates the host response to L. pneumophila infection in a manner similar to that of murine Naip5 and that human NAIP and murine Naip5 regulate cell survival by inhibiting apoptosis or by promoting pyroptosis in response to specific cellular signals.

Regulation of osteoclastogenesis by simon extracts composed of caffeic acid and related compounds : successful suppression of bone destruction accompanied with adjuvant-induced arthritis in rats

Simon extracts are vitamin K1-rich food materials extracted from the leaves of the Simon sweet potato. Although vitamin K is known to stimulate bone formation, we postulated that Simon extracts also contain unknown biological compounds having the ability to regulate bone resorption. Here we prepared the vitamin K-free fraction from the Simon extracts and investigated the ability of this fraction on the differentiation of osteoclasts. A remarkable inhibitory effect of osteoclastogenesis was observed when osteoclast precursors were treated with this fraction in rat bone marrow culture systems as well as in a pure differentiation system using murine osteoclast precursor cell line. The vitamin K-free Simon extracts markedly suppressed severe bone destruction mediated by abundant osteoclasts associated with adjuvant-induced arthritis in rats. High performance liquid chromatography (HPLC) analysis revealed that the vitamin K-free Simon extracts contained three types of low molecular weight inhibitors for osteoclastogenesis; caffeic acid, chlorogenic acids and isochlorogenic acids. Among these substances, caffeic acid showed the most powerful inhibitory effects on osteoclastogenesis. Caffeic acid significantly suppressed expression of NFATc1, a key transcription factor for the induction of osteoclastogenesis. Our current study enlightened a high utility of the Simon extracts and their chemical components as effective regulators for bone resorption accompanied with inflammation and metabolic bone diseases.