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Sakae Tanaka

Publications and source records attributed to Sakae Tanaka.

At least 37 records · Page 2Linked to original sources
PubMed2005

In vitro and in vivo assays for osteoclast apoptosis.

Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone.

Journal Article↗
PubMed2005

RANK-mediated amplification of TRAF6 signaling leads to NFATc1 induction during osteoclastogenesis.

RANK and CD40 activate NF-kappaB and MAPKs to similar levels via TRAF6. Even though overexpression of TRAF6 results in osteoclast formation, RANK but not CD40 promotes osteoclastogenesis. To understand the molecular basis for RANK-specific activity in osteoclastogenesis, we created an osteoclast formation system driven by anti-human CD40 antibody-mediated stimulation of a chimeric receptor, h40/mRK, which consists of the extracellular domain of human CD40 and the transmembrane and cytoplasmic domains of mouse RANK. By introducing mutations into three TRAF6-binding sites of RANK, we found that h40/mRK with a single TRAF6-binding site efficiently induced Ca2+ oscillation and expression of NFATc1, a master switch in osteoclastogenesis, whereas CD40 carrying a single TRAF6-binding site did not. However, expression of CD40 that was approximately 100 times greater than that of h40/mRK resulted in osteoclast formation, indicating that the RANK-TRAF6 signal is more potent than the CD40-TRAF6 signal in terms of NFATc1 activation and osteoclastogenesis. These results suggest that RANK may harbor a specific domain that amplifies TRAF6 signaling.

Animals↗
PubMed2005

Intracellular signal transduction pathways: good therapeutic targets for joint destruction in rheumatoid arthritis.

Preventing joint destruction is one of the most challenging issues in treating patients with rheumatoid arthritis (RA), and I propose that intracellular signaling pathways in osteoclasts and synovial fibroblastic cells (SFCs) can be good therapeutic targets. Osteoclasts are primarily involved in the bone destruction in RA joints, and SFCs support osteoclast differentiation and activation by producing various proinflammatory cytokines including receptor activator of NF-kappaB ligand (RANKL), the osteoclast differentiation factor belonging to the tumor necrosis factor-alpha superfamily. Suppressing c-Src pathways by adenovirus vector-mediated C-terminal Src family kinase (Csk) gene or Ras/extracellular-regulating kinase (ERK) pathways by introducing dominant negative Ras (Ras(DN)) adenovirus reduced osteoclastic bone resorption as well as the abnormal proliferation and interleukin-6 production of SFCs, and the local injection of these viruses ameliorated the joint destruction in adjuvant arthritis rats. Moreover, chondrogenic differentiation of SFCs could be induced by stimulating activin receptor-like kinase 3 pathways.

Journal Article↗
PubMed2005

Role of RANKL in physiological and pathological bone resorption and therapeutics targeting the RANKL-RANK signaling system.

Osteoclasts are primary cells for physiological and pathological bone resorption, and receptor activator of nuclear factor-kappaB ligand (RANKL) is critically involved in the differentiation, activation, and survival of these cells. Recently, therapeutics for pathological bone destruction targeting RANKL pathways has attracted a great deal of attention. Herein, we review the recent advances in the research on osteoclast biology and discuss the advantages and disadvantages of anti-RANKL therapies.

Animals↗
PubMed2005

Integrin alpha4beta1 promotes focal adhesion kinase-independent cell motility via alpha4 cytoplasmic domain-specific activation of c-Src.

The fibronectin binding integrins alpha5beta1 and alpha4beta1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For alpha5beta1, beta1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for alpha5beta1-stimulated cell motility and that exogenous expression of human alpha4 in FAK-null fibroblasts forms a functional alpha4beta1 receptor that promotes robust cell motility equal to the alpha5beta1 stimulation of wild-type and FAK-reconstituted fibroblasts. alpha4beta1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the alpha4 cytoplasmic domain, independent of direct paxillin binding to alpha4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. alpha4 cytoplasmic domain-initiated signaling led to a approximately 4-fold activation of c-Src which did not require paxillin binding to alpha4. Notably, alpha4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase alpha overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. alpha4beta1-stimulated cell motility of triple-null Src(-/-), c-Yes(-/-), and Fyn(-/-) fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for alpha5beta1-stimulated FAK activation, our results support the existence of a novel alpha4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway.

Amino Acid Sequence↗
PubMed2005

[Disorder of bone quality in osteopetrosis].

Osteopetrosis is a genetic disorder characterized by increased bone mass due to the reduced bone resorption. It is also characterized by the discrepancy between bone mass and bone strength. In this review, I would like to introduce the up-to-date of the research and treatment of osteopetrosis.

Adult↗
PubMed2005

[A novel therapeutic vaccine approach against RANKL that prevents pathological bone destruction].

The vaccination method is used to induce antibody response against disease-related proteins by immunization with modified pathological antigen as preventive therapy to acquire disease resistance. A RANKL vaccine was developed by modifying the soluble TNF-like domain of murine RANKL to incorporate a promiscuous Th epitope. Immunization with RANKL vaccine prevented pathological bone resorption in arthritic mice or ovariactomized mice.

Animals↗
PubMed2005

[Mechanism of the bone destruction in rheumatoid arthritis].

Rheumatoid arthritis (RA) is characterized by the presence of inflammatory synovitis accompanied by cartilage and bone destruction. Histological examination of RA pannus shows a number of osteoclasts on the surface of the destructed bone. RA synovial tissues produce a variety of proinflammatory cytokines and growth factors that may increase osteoclast formation, activity, and/or survival. Synovial fibroblasts from RA patients express high levels of RANKL, which is essential for the differentiation of osteoclasts, and therefore, RANKL can be a good therapeutic target of joint destruction in RA.

Arthritis, Rheumatoid↗
PubMed2005

[RANKL vaccination].

A therapeutic vaccine approach, targeting RANKL, can be used to inhibit bone destruction in pathological bone loss conditions such as osteoporosis, rheumatoid arthritis and bone metastasis. Compared to other therapeutic recombinant protein against pathological antigen such as monoclonal antibodies, soluble receptor or other antagonists, a vaccine needs small doses of protein to induce its affect. Once the immune response is established by vaccination, it is maintained by boosting with the vaccine. This RANKL vaccination would be a novel approach to any kind of pathological bone

Animals↗
PubMed2004

Essential role of synoviolin in embryogenesis.

We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect. For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno(-/-)) mice by genetargeted disruption. Strikingly, all fetuses lacking syno died in utero around embryonic day 13.5, although Hrd1p, a yeast orthologue of Synoviolin, is non-essential for survival. Histologically, hypocellularity and aberrant apoptosis were noted in the syno(-/-) fetal liver. Moreover, definitive erythropoiesis was affected in non-cell autonomous manner in syno(-/-) embryos, causing death in utero. Cultured embryonic fibroblasts derived from syno(-/-) mice were more susceptible to endoplasmic reticulum stress-induced apoptosis than those from syno(+/+) mice, but the susceptibility was rescued by overexpression of synoviolin. Our findings emphasized the indispensable role of the Synoviolin in embryogenesis.

Adenoviridae↗
PubMed2004

Monokine induced by interferon-gamma is induced by receptor activator of nuclear factor kappa B ligand and is involved in osteoclast adhesion and migration.

Bone remodeling is accompanied by the differentiation of osteoclasts from the monocyte/macrophage lineage of hematopoietic cells. The osteoclast differentiation process requires receptor activator of nuclear factor kappa B (NF-kappa B) ligand (RANKL), which causes complex changes in the expression of various genes. In a cDNA microarray study to identify genes targeted by RANKL, we found that monokine induced by the interferon-gamma (IFN-gamma) (MIG) gene was up-regulated in osteoclast precursor cells. The increase in MIG expression by RANKL was confirmed by reverse transcription-polymerase chain reaction and Western blot analysis. RANKL induction of MIG required the activity of NF-kappa B, whose binding site is present in the MIG promoter. MIG induction by RANKL was also dependent on p38 mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 1 (STAT1). RANKL stimulated the phosphorylation of Ser727 of STAT1, which required p38 activity. MIG secreted on RANKL treatment could stimulate the migration and adhesion of osteoclast precursors and osteoclasts that were primed to express CXCR3, the MIG receptor, by macrophage-colony-stimulating factor (M-CSF). Therefore, we provide the first evidence demonstrating that RANKL stimulates the serine phosphorylation of STAT1 through the p38 MAPK pathway, causing MIG gene transcription and secretion, which may have a role in recruiting CXCR3-positive osteoclast precursors and osteoclasts to bone remodeling or inflammatory sites.

Animals↗
PubMed2004

Opposing extracellular signal-regulated kinase and Akt pathways control Schwann cell myelination.

Schwann cells are the myelinating glia of the peripheral nervous system, and their development is regulated by various growth factors, such as neuregulin, platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). However, the mechanism of intracellular signaling pathways following these ligand stimuli in Schwann cell differentiation remains elusive. Here, we demonstrate that in cultured Schwann cells, neuregulin and PDGF suppressed the expression of myelin-associated protein markers, whereas IGF-I promoted it. Although these ligands activated common downstream signaling pathways [i.e., extracellular signal-regulated kinase (Erk) and phosphatidylinositol-3-kinase (PI3K)-Akt pathways], the profiles of activation varied among ligands. To elucidate the function of these pathways and the mechanisms underlying Schwann cell differentiation, we used adenoviral vectors to selectively activate or inactivate these pathways. We found that the selective activation of Erk pathways suppressed Schwann cell differentiation, whereas that of PI3K pathways promoted it. Furthermore, lithium chloride, a modulator of glycogen synthase kinase-3beta (GSK-3beta) promoted Schwann cell differentiation, suggesting the involvement of GSK-3beta as a downstream molecule of PI3K-Akt pathways. Selective activation of PI3K pathways in Schwann cells by gene transfer also demonstrated increased myelination in in vitro Schwann cell-DRG neuron cocultures and in vivo allogenic nerve graft experiments. We conclude that signals mediated by PI3K-Akt are crucial for initiation of myelination and that the effects of growth factors are primarily dependent on the balance between Erk and PI3K-Akt activation. Our results also propose the possibility of augmenting Schwann cell functions by modulating intracellular signals in light of future cell therapies.

Animals↗
PubMed2004

Osteoclast differentiation by RANKL requires NF-kappaB-mediated downregulation of cyclin-dependent kinase 6 (Cdk6).

UNLABELLED: This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-kappaB signaling. INTRODUCTION: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. MATERIALS AND METHODS: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-kappaB and c-jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the IkappaB kinase 2 (IKK(DN)) gene and mitogen-activated protein kinase kinase 7 (MKK7(DN)) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. RESULTS AND CONCLUSION: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-kappaB pathway by IKK(DN) overexpression, but not that of the JNK pathway by MKK7(DN) overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-kappaB-mediated downregulation is essential for efficient osteoclast differentiation.

Animals↗
PubMed2004

Fcgamma receptor signaling in primary human microglia: differential roles of PI-3K and Ras/ERK MAPK pathways in phagocytosis and chemokine induction.

Cryptococcus neoformans monoclonal antibody immune complex (IC) induces beta-chemokines and phagocytosis in primary human microglia via activation of Fc receptor for immunoglobulin G (FcgammaR). In this report, we investigated microglial FcgammaR signal-transduction pathways by using adenoviral-mediated gene transfer and specific inhibitors of cell-signaling pathways. We found that Src inhibitor PP2 and Syk inhibitor piceatannol inhibited phagocytosis, macrophage-inflammatory protein-1alpha (MIP-1alpha) release, as well as phosphorylation of extracellular-regulated kinase (ERK) and Akt, consistent with Src/Syk involvement early in FcgammaR signaling. Constitutively active mitogen-activated protein kinase kinase (MEK) induced MIP-1alpha, and Ras dominant-negative (DN) inhibited IC-induced ERK phosphorylation and MIP-1alpha production. These results suggest that the Ras/MEK/ERK pathway is necessary and sufficient in IC-induced MIP-1alpha expression. Neither Ras DN nor the MEK inhibitor U0126 inhibited phagocytosis. In contrast, phosphatidylinositol-3 kinase (PI-3K) inhibitors Wortmannin and LY294002 inhibited phagocytosis without affecting ERK phosphorylation or MIP-1alpha production. Conversely, Ras DN or U0126 did not affect Akt phosphorylation. Together, these results demonstrate distinct roles played by the PI-3K and Ras/MEK/ERK pathways in phagocytosis and MIP-1alpha induction, respectively. Our results demonstrating activation of functionally distinct pathways following microglial FcgammaR engagement may have implications for human central nervous system diseases.

Adenoviridae↗
PubMed2004

Src kinase activity is essential for osteoclast function.

Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.

Adenoviridae↗
PubMed2004

Suppression of T cell responses by chondromodulin I, a cartilage-derived angiogenesis inhibitory factor: therapeutic potential in rheumatoid arthritis.

OBJECTIVE: Chondromodulin I (ChM-I), a cartilage matrix protein, promotes the growth and proteoglycan synthesis of chondrocytes. However, it also inhibits angiogenesis. Since ChM-I is expressed not only in cartilage, but also in the thymus, we investigated the modulation of T cell function by ChM-I to assess its therapeutic potential in rheumatoid arthritis (RA). METHODS: The localization of ChM-I expression in mouse thymus tissue was examined by in situ hybridization. The proliferative response of peripheral blood T cells and synovial cells obtained from patients with RA was evaluated by (3)H-thymidine incorporation assay. The effects of ChM-I were examined using recombinant human ChM-I (rHuChM-I). Modulation of the antigen-specific immune response was evaluated by the recall response of splenic T cells and the delayed-type hypersensitivity response induced in the ear of mice primed with ovalbumin (OVA). Antigen-induced arthritis (AIA) was induced in mice by injecting methylated bovine serum albumin into the ankle joints 2 weeks after the priming. RESULTS: ChM-I was expressed in the cortex of the thymus. Recombinant human ChM-I suppressed the proliferative response of mouse splenic T cells and human peripheral blood T cells stimulated with anti-CD3/CD28 antibodies, in a dose-dependent manner. Production of interleukin-2 was decreased in rHuChM-I-treated mouse CD4 T cells. Ten micrograms of rHuChM-I injected intraperitoneally into OVA-primed mice suppressed the induction of the antigen-specific immune response. Finally, rHuChM-I suppressed the development of AIA, and also suppressed the proliferation of synovial cells prepared from the joints of patients with RA. CONCLUSION: These results suggest that ChM-I suppresses T cell responses and synovial cell proliferation, implying that this cartilage matrix protein has a therapeutic potential in RA.

Adjuvants, Immunologic↗
PubMed2004

Traf6 is essential for murine tooth cusp morphogenesis.

Ectodermal appendages such as skin, hair, teeth, and sweat glands are affected in patients with hypohidrotic (anhydrotic) ectodermal dysplasia (HED). It has been established that mutations in the tumor necrosis factor (TNF) superfamily of molecules, i.e., ectodysplasin (EDA), EDA receptor (EDAR), and EDAR-associated death domain (EDARADD; the intracellular adaptor for EDAR), are responsible for several forms of HED in humans and mice. We show here by in situ hybridisation that another TNF family (orphan) receptor, TROY (also known TAJ, TAJ-alpha, TRADE, and TNFRSF19), is strongly coexpressed with Edar in the epithelial enamel knot signalling centres that are believe to regulate cuspal morphogenesis during murine tooth development. Traf6 is known to function as an intracellular adaptor protein for Troy and examination of Traf6 mutant mice revealed abnormalities in molar teeth that are similar but more severe than those produced by mutations in Eda signalling molecules. This finding suggests that, in additional to ectodysplasin, another TNF pathway involving Troy/Traf6 is involved in molar tooth cusp formation and identifies an essential role for a Traf in tooth development. Developmental Dynamics 229:131-135, 2004.

Animals↗