Biomedical subjects

Takako Koga

Publications and source records attributed to Takako Koga.

13 recordsLinked to original sources

PubMed2005

NFAT and Osterix cooperatively regulate bone formation.

Immunosuppressants are crucial in the prevention of detrimental immune reactions associated with allogenic organ transplantation, but they often cause adverse effects in a number of biological systems, including the skeletal system. Calcineurin inhibitors FK506 and cyclosporin A inhibit nuclear factor of activated T cells (NFAT) activity and induce strong immunosuppression. Among NFAT proteins, NFATc1 is crucial for the differentiation of bone-resorbing osteoclasts. Here we show FK506 administration induces the reduction of bone mass despite a blockade of osteoclast differentiation. This reduction is caused by severe impairment of bone formation, suggesting that NFAT transcription factors also have an important role in the transcriptional program of osteoblasts. In fact, bone formation is inhibited in Nfatc1- and Nfatc2-deficient cells as well as in FK506-treated osteoblasts. Overexpression of NFATc1 stimulates Osterix-dependent activation of the Col1a1 (encoding type I collagen) promoter, but not Runx2-dependent activation of the Bglap1 (encoding osteocalcin) promoter. NFAT and Osterix form a complex that binds to DNA, and this interaction is important for the transcriptional activity of Osterix. Thus, NFAT and Osterix cooperatively control osteoblastic bone formation. These results may provide important insight into the management of post-transplantation osteoporosis as well as a new strategy for promoting bone regeneration in osteopenic disease.

Animals↗

PubMed2005

Stat1-mediated cytoplasmic attenuation in osteoimmunology.

Signal transducer and activator of transcription 1 (Stat1) is a critical mediator of gene transcription in type I interferon (IFN-alpha/beta) signaling that is essential for host defense against viruses. In the skeletal system, type I IFNs (IFN-alpha/beta) also play an important physiological role in the inhibition of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation and bone resorption: mice deficient in IFN signaling exhibit decreased bone mass accompanied by the activation of osteoclastogenesis. On the other hand, an unexpected increase in bone mass was observed in Stat1-deficient mice, indicating that Stat1 has a hitherto unknown function in the regulation of bone formation. Indeed, Stat1 was found to have a unique, non-canonical function as a cytoplasmic attenuator of Runx2, a key transcription factor for osteoblast differentiation. Thus, the loss of Stat1 results in excessive activation of Runx2 and osteoblast differentiation, thereby tipping the balance in favor of bone formation over bone resorption. This is an interesting example in which a latent transcription factor attenuates the activity of another transcription factor in the cytoplasm, and reveals a novel regulatory mechanism of bone remodeling by immunomodulatory molecules. Here, we summarize recent advances in the study of Stat1 and IFNs in the context of osteoimmunology, including latest reports that question whether the inhibitory function of Stat1 in chondrocytes is responsible for dwarfism in achondroplasia.

Animals↗

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↗

PubMed2004

Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis.

Costimulatory signals are required for activation of immune cells, but it is not known whether they contribute to other biological systems. The development and homeostasis of the skeletal system depend on the balance between bone formation and resorption. Receptor activator of NF-kappaB ligand (RANKL) regulates the differentiation of bone-resorbing cells, osteoclasts, in the presence of macrophage-colony stimulating factor (M-CSF). But it remains unclear how RANKL activates the calcium signals that lead to induction of nuclear factor of activated T cells c1, a key transcription factor for osteoclastogenesis. Here we show that mice lacking immunoreceptor tyrosine-based activation motif (ITAM)-harbouring adaptors, Fc receptor common gamma subunit (FcRgamma) and DNAX-activating protein (DAP)12, exhibit severe osteopetrosis owing to impaired osteoclast differentiation. In osteoclast precursor cells, FcRgamma and DAP12 associate with multiple immunoreceptors and activate calcium signalling through phospholipase Cgamma. Thus, ITAM-dependent costimulatory signals activated by multiple immunoreceptors are essential for the maintenance of bone homeostasis. These results reveal that RANKL and M-CSF are not sufficient to activate the signals required for osteoclastogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2004

The antirheumatic drug leflunomide inhibits osteoclastogenesis by interfering with receptor activator of NF-kappa B ligand-stimulated induction of nuclear factor of activated T cells c1.

OBJECTIVE: Suppression of bone destruction is required as part of an effective therapeutic strategy for autoimmune arthritis. Although numerous antirheumatic drugs are in clinical use, little is known about whether they inhibit bone destruction by acting on activated T cells or other cell types, such as bone-resorbing osteoclasts. This study was undertaken to determine whether leflunomide has a direct action on the osteoclast lineage and to gain insights into the molecular basis for the bone-protective effect of leflunomide. METHODS: The direct effect of leflunomide on osteoclast differentiation was investigated using an in vitro culture system of bone marrow monocyte/macrophages stimulated with receptor activator of NF-kappa B ligand (RANKL) and macrophage colony-stimulating factor. The molecular mechanism of the inhibition was analyzed by genome-wide screening. The T cell-independent effect of leflunomide was examined in rag-2(-/-) mice. RESULTS: Leflunomide blocked de novo pyrimidine synthesis and RANKL-induced calcium signaling in osteoclast precursor cells in vitro; hence, the induction of nuclear factor of activated T cells c1 (NF-ATc1) was strongly inhibited. The inhibition of this pathway is central to the action of leflunomide, since the inhibition was overcome by ectopic expression of NF-ATc1 in the precursor cells. Leflunomide suppressed endotoxin-induced inflammatory bone destruction even in rag-2(-/-) mice. CONCLUSION: Leflunomide has a direct inhibitory effect on RANKL-mediated osteoclast differentiation by inhibiting the induction of NF-ATc1, the master switch regulator for osteoclast differentiation. Our study suggests that the direct inhibitory action of leflunomide on osteoclast differentiation constitutes an important aspect in the amelioration of bone destruction, and that the RANKL-dependent NF-ATc1 induction pathway is a promising target for pharmacologic intervention in arthritic bone destruction.

Animals↗

PubMed2004

Sorting nexin homologues are targets of phosphatidylinositol 3-phosphate in sporulation of Schizosaccharomyces pombe.

Schizosaccharomyces pombe defective in phosphatidylinositol (PtdIns) 3-kinase shows various defects in forespore membrane formation, including onset, growth orientation, and closure. Downstream factors of PtdIns 3-kinase in this system were explored. Among various phox homology (PX) domain-containing proteins, Vps5p and Vps17p, homologues of sorting nexins, were found to be required for efficient sporulation. Cells defective in these proteins showed a disordered growth orientation of the forespore membrane, as is the case with Deltapik3 cells. Vps5p and Vps17p with mutations in the PX domains failed to suppress the defects of their relevant disruptants. Vps5p and Vps17p migrated toward the the forespore membrane in a pik3+-dependent manner, suggesting that these proteins may interact with PtdIns(3)P. Electron-microscopic analysis revealed that the forespore membrane fails to engulf the nucleus in some of these cells, accumulating vesicle-like bodies similar to those seen in Deltaspo3 cells. These results suggest that Vps5p and Vps17p are the targets of PtdIns(3)P in vesicle transport required for onset of the forespore membrane formation.

Amino Acid Sequence↗

PubMed2003

Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation.

Bone remodeling is central to maintaining the integrity of the skeletal system, wherein the developed bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic bone resorption. In the present study, we demonstrate a novel function of the Stat1 transcription factor in the regulation of bone remodeling. In the bone of the Stat1-deficient mice, excessive osteoclastogenesis is observed, presumably caused by a loss of negative regulation of osteoclast differentiation by interferon (IFN)-beta. However, the bone mass is unexpectedly increased in these mice. This increase is caused by excessive osteoblast differentiation, wherein Stat1 function is independent of IFN signaling. Actually, Stat1 interacts with Runx2 in its latent form in the cytoplasm, thereby inhibiting the nuclear localization of Runx2, an essential transcription factor for osteoblast differentiation. The new function of Stat1 does not require the Tyr 701 that is phosphorylated when Stat1 becomes a transcriptional activator. Our study provides a unique example in which a latent transcription factor attenuates the activity of another transcription factor in the cytoplasm, and reveals a new regulatory mechanism in bone remodeling.

Animals↗

PubMed2003

Characterization of vps33+, a gene required for vacuolar biogenesis and protein sorting in Schizosaccharomyces pombe.

From the fission yeast Schizosaccharomyces pombe we have identified and deleted vps33, a gene encoding a homologue of VPS33, which is required for vacuolar biogenesis in S. cerevisiae cells. When the vps33(+) gene is disrupted, Sz. pombe strains are temperature-sensitive for growth and contain numerous small vesicular structures stained with FM4-64 in the cells. Deletion of the Sz. pombe vps33(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and sporulation defects. These results are consistent with Vps33p being necessary for the morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz. pombe cells.

Amino Acid Sequence↗

PubMed2003

Role of phosphatidylinositol 3-phosphate in formation of forespore membrane in Schizosaccharomyces pombe.

Phosphatidylinositol (PI) 3-kinase (encoded by the pik3(+) gene) in Schizosaccharomyces pombe has been identified as a homologue of VPS34p, a protein required for proper vesicular protein sorting. The clone defective in this protein carries enlarged vacuoles and exhibits sensitivity to high temperature or high ion concentration. The effect of disruption of pik3(+) on sporulation of Sz. pombe was examined. The diploid cells underwent G(1) arrest and meiosis. However, the spores formed by the deltapik3 cells were not viable. Electron-microscopic analysis revealed that the growth of the forespore membrane of deltapik3 cells was not correctly orientated, failing to engulf the nucleus or forming extremely small spores, as was confirmed by the use of Spo3p-GFP and GFP-Psy1p, which are markers of the forespore membrane. The coating materials found along the forespore membrane of the wild-type were greatly reduced in these cells. PI 3-P, the product of Pik3p, was detected on the forespore membrane, suggesting that PI 3-P-dependent vesicle transport may take place in formation of the forespore membrane. Misshaped forespore membrane, accumulation of vesicles, formation of small non-viable spores, and suppression by over expression of Psy1p were the phenotypes commonly seen in deltapik3 and deltaspo3 cells, suggesting a relationship between the functions of Pik3p and Spo3p in formation of the forespore membrane in Sz. pombe.

Cell Membrane↗

PubMed2003

Inportance of phosphatidylinositol 3-phosphate in sporulation of Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, Pik3p phosphorylates phosphatidylinositol (PI) to produce PI 3-P, which is further phosphorylated by Ste12p to yield PI 3,5-P2. Pik3p is required for both conjugation and sporulation. To test which of PI 3-P and PI 3,5-P2 is required for sporulation, diploid cells defective in production of PI 3,5-P2 were used. They underwent sporulation almost normally provided that the osmotic pressure of the medium was controlled, suggesting that not PI 3,5-P2 but PI 3-P was important. Electron microscopic analysis confirmed normal sporulation in the absence of PI 3,5-P2 although the forespore membrane was found to be less dense in these cells.

Culture Media↗

PubMed2003

Isolation of suppressor mutants of phosphatidylinositol 3-phosphate 5-kinase deficient cells in Schizosaccharomyces pombe.

The ste12+ gene of Schizosaccharomyces pombe codes for a phosphatidylinositol (PI) 3-phosphate 5'-kinase, which is required for efficient mating. Suppressor mutants for sterility of ste12Delta cells were screened for. Most of the mutant genes turned out to be recessive. Six genes were cloned and the open reading frames responsible for the suppressor activity were identified. They included genes coding for proteins with domains homologous to calcium transporters, casein kinase II, UBC13, AMSH, Vps23p, and Vps27p of Saccharomyces cerevisiae. Disruption of these genes resulted in suppression of the defects of the ste12Delta cells, including low mating efficiency and formation of large vacuoles. Since many of these gene products are homologous to the proteins involved in vesicle transport, sterility caused by inactivation of ste12 may be due to a disordered vesicle transport system.

Amino Acid Sequence↗

PubMed2002

Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts.

Signaling by RANKL is essential for terminal differentiation of monocytes/macrophages into osteoclasts. The TRAF6 and c-Fos signaling pathways both play important roles downstream of RANKL. We show here that RANKL selectively induces NFATc1 expression via these two pathways. RANKL also evokes Ca(2+) oscillations that lead to calcineurin-mediated activation of NFATc1, and therefore triggers a sustained NFATc1-dependent transcriptional program during osteoclast differentiation. We also show that NFATc1-deficient embryonic stem cells fail to differentiate into osteoclasts in response to RANKL stimulation, and that ectopic expression of NFATc1 causes precursor cells to undergo efficient differentiation without RANKL signaling. Thus, NFATc1 may represent a master switch for regulating terminal differentiation of osteoclasts, functioning downstream of RANKL.

Active Transport, Cell Nucleus↗

PubMed2002

Phosphatidylinositol 3-phosphate 5-kinase is required for the cellular response to nutritional starvation and mating pheromone signals in Schizosaccharomyces pombe.

BACKGROUND: Phosphatidylinositol (3,5) bisphosphate, which is converted from phosphatidylinositol 3-phosphate by phosphatidylinositol 3-phosphate 5-kinase, is implicated in vacuolar functions and the sorting of cell surface proteins within endosomes in the endocytic pathway of budding yeast. A homologous protein, SpFab1p, has been found in the fission yeast Schizosaccharomyces pombe, but its role is not known. RESULTS: Here we report that SpFab1p is encoded by ste12+ known as a fertility gene in S. pombe. The ste12 mutant grew normally under stress-free conditions, but was highly vacuolated and swelled at high temperatures and under starvation conditions. In nitrogen-free medium, ste12 cells were arrested in G1 phase, but partially defective in the expression of genes responsible for mating and meiosis. The ste12 mutant was defective both in the production of, and in the response to, mating pheromones. The amount of the pheromone receptor protein Map3p, was substantially decreased in ste12 cells. Map3p was transported to the cell surface, then internalized and eventually transported to the vacuolar lumen, even in the ste12 mutant. CONCLUSION: The results indicate that phosphatidylinositol(3,5)bisphosphate is essential for cellular responses to various stresses and for the mating pheromone signalling under starvation conditions.

Amino Acid Sequence↗