Browsing by Author "Ueki, Yasuyoshi"
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Item Alveolar bone protection by targeting the SH3BP2-SYK axis in osteoclasts(Wiley, 2020-02) Kittaka, Mizuho; Yoshimoto, Tetsuya; Schlosser, Collin; Rottapel, Robert; Kajiya, Mikihito; Kurihara, Hidemi; Reichenberger, Ernst J.; Ueki, Yasuyoshi; Biomedical Sciences and Comprehensive Care, School of DentistryPeriodontitis is a bacterially induced chronic inflammatory condition of the oral cavity where tooth-supporting tissues including alveolar bone are destructed. Previously, we have shown that the adaptor protein SH3-domain binding protein 2 (SH3BP2) plays a critical role in inflammatory response and osteoclastogenesis of myeloid lineage cells through spleen tyrosine kinase (SYK). In this study, we show that SH3BP2 is a novel regulator for alveolar bone resorption in periodontitis. Micro-CT analysis of SH3BP2-deficient (Sh3bp2 -/- ) mice challenged with ligature-induced periodontitis revealed that Sh3bp2 -/- mice develop decreased alveolar bone loss (male 14.9% ± 10.2%; female 19.0% ± 6.0%) compared with wild-type control mice (male 25.3% ± 5.8%; female 30.8% ± 5.8%). Lack of SH3BP2 did not change the inflammatory cytokine expression and osteoclast induction. Conditional knockout of SH3BP2 and SYK in myeloid lineage cells with LysM-Cre mice recapitulated the reduced bone loss without affecting both inflammatory cytokine expression and osteoclast induction, suggesting that the SH3BP2-SYK axis plays a key role in regulating alveolar bone loss by mechanisms that regulate the bone-resorbing function of osteoclasts rather than differentiation. Administration of a new SYK inhibitor GS-9973 before or after periodontitis induction reduced bone resorption without affecting inflammatory reaction in gingival tissues. In vitro, GS-9973 treatment of bone marrow-derived M-CSF-dependent macrophages suppressed tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation with decreased mineral resorption capacity even when GS-9973 was added after RANKL stimulation. Thus, the data suggest that SH3BP2-SYK is a novel signaling axis for regulating alveolar bone loss in periodontitis and that SYK can be a potential therapeutic target to suppress alveolar bone resorption in periodontal diseases.Item Editorial: A new frontier in translational research on autoinflammatory diseases - various aspects of innate immunity on human diseases(Frontiers Media, 2023-01-31) Mukai, Tomoyuki; Ida, Hiroaki; Ueki, Yasuyoshi; Nishikomori, Ryuta; Biomedical Sciences and Comprehensive Care, School of DentistryItem Imatinib has minimal effects on inflammatory and osteopenic phenotypes in a murine cherubism model(Wiley, 2021) Mukai, Tomoyuki; Akagi, Takahiko; Hiramatsu Asano, Sumie; Tosa, Ikue; Ono, Mitsuaki; Kittaka, Mizuho; Ueki, Yasuyoshi; Yahagi, Ayano; Iseki, Masanori; Oohashi, Toshitaka; Ishihara, Katsuhiko; Morita, Yoshitaka; Biomedical Sciences and Comprehensive Care, School of DentistryObjective Cherubism is a genetic disorder characterised by bilateral jawbone deformation. The associated jawbone lesions regress after puberty, whereas severe cases require surgical treatment. Although several drugs have been tested, fundamental treatment strategies for cherubism have not been established. The effectiveness of imatinib has recently been reported; however, its pharmaceutical mechanism remains unclear. In this study, we tested the effects of imatinib using a cherubism mouse model. Methods We used Sh3bp2 P416R cherubism mutant mice, which exhibit systemic organ inflammation and osteopenia. The effects of imatinib were determined using primary bone marrow-derived macrophages. Imatinib was administered intraperitoneally to the mice, and serum tumour necrosis factor-α (TNFα), organ inflammation and bone properties were examined. Results The cherubism mutant macrophages produced higher levels of TNFα in response to lipopolysaccharide compared to wild-type macrophages, and imatinib did not significantly suppress TNFα production. Although imatinib suppressed osteoclast formation in vitro, administering it in vivo did not suppress organ inflammation and osteopenia. Conclusion The in vivo administration of imatinib had a minimal therapeutic impact in cherubism mutant mice. To establish better pharmaceutical interventions, it is necessary to integrate new findings from murine models with clinical data from patients with a definitive diagnosis of cherubism.Item Inhibition of RANKL improves the skeletal phenotype of adenine-induced chronic kidney disease in mice(Oxford University Press, 2024-01-14) Metzger, Corinne E.; Kittaka, Mizuho; LaPlant, Alec N.; Ueki, Yasuyoshi; Allen, Matthew R.; Anatomy, Cell Biology and Physiology, School of MedicineSkeletal fragility and high fracture rates are common in CKD. A key component of bone loss in CKD with secondary hyperparathyroidism is high bone turnover and cortical bone deterioration through both cortical porosity and cortical thinning. We hypothesized that RANKL drives high bone resorption within cortical bone leading to the development of cortical porosity in CKD (study 1) and that systemic inhibition of RANKL would mitigate the skeletal phenotype of CKD (study 2). In study 1, we assessed the skeletal properties of male and female Dmp1-cre RANKLfl/fl (cKO) and control genotype (Ranklfl/fl; Con) mice after 10 wk of adenine-induced CKD (AD; 0.2% dietary adenine). All AD mice regardless of sex or genotype had elevated blood urea nitrogen and high PTH. Con AD mice in both sexes had cortical porosity and lower cortical thickness as well as high osteoclast-covered trabecular surfaces and higher bone formation rate. cKO mice had preserved cortical bone microarchitecture despite high circulating PTH as well as no CKD-induced increases in osteoclasts. In study 2, male mice with established AD CKD were either given a single injection of an anti-RANKL antibody (5 mg/kg) 8 wk post-induction of CKD or subjected to 3×/wk dosing with risedronate (1.2 μg/kg) for 4 wk. Anti-RANKL treatment significantly reduced bone formation rate as well as osteoclast surfaces at both trabecular and cortical pore surfaces; risedronate treatment had little effect on these bone parameters. In conclusion, these studies demonstrate that bone-specific RANKL is critical for the development of high bone formation/high osteoclasts and cortical bone loss in CKD with high PTH. Additionally, systemic anti-RANKL ligand therapy in established CKD may help prevent the propagation of cortical bone loss via suppression of bone turnover.Item Loss-of-function OGFRL1 variants identified in autosomal recessive cherubism families(Oxford University Press, 2024-04-09) Kittaka, Mizuho; Mizuno, Noriyoshi; Morino, Hiroyuki; Yoshimoto, Tetsuya; Zhu, Tianli; Liu, Sheng; Wang, Ziyi; Mayahara, Kotoe; Iio, Kyohei; Kondo, Kaori; Kondo, Toshio; Hayashi, Tatsuhide; Coghlan, Sarah; Teno, Yayoi; Doan, Andrew Anh Phung; Levitan, Marcus; Choi, Roy B.; Matsuda, Shinji; Ouhara, Kazuhisa; Wan, Jun; Cassidy, Annelise M.; Pelletier, Stephane; Nampoothiri, Sheela; Urtizberea, Andoni J.; Robling, Alexander G.; Ono, Mitsuaki; Kawakami, Hideshi; Reichenberger, Ernst J.; Ueki, Yasuyoshi; Anatomy, Cell Biology and Physiology, School of MedicineCherubism (OMIM 118400) is a rare craniofacial disorder in children characterized by destructive jawbone expansion due to the growth of inflammatory fibrous lesions. Our previous studies have shown that gain-of-function mutations in SH3 domain-binding protein 2 (SH3BP2) are responsible for cherubism and that a knock-in mouse model for cherubism recapitulates the features of cherubism, such as increased osteoclast formation and jawbone destruction. To date, SH3BP2 is the only gene identified to be responsible for cherubism. Since not all patients clinically diagnosed with cherubism had mutations in SH3BP2, we hypothesized that there may be novel cherubism genes and that these genes may play a role in jawbone homeostasis. Here, using whole exome sequencing, we identified homozygous loss-of-function variants in the opioid growth factor receptor like 1 (OGFRL1) gene in 2 independent autosomal recessive cherubism families from Syria and India. The newly identified pathogenic homozygous variants were not reported in any variant databases, suggesting that OGFRL1 is a novel gene responsible for cherubism. Single cell analysis of mouse jawbone tissue revealed that Ogfrl1 is highly expressed in myeloid lineage cells. We generated OGFRL1 knockout mice and mice carrying the Syrian frameshift mutation to understand the in vivo role of OGFRL1. However, neither mouse model recapitulated human cherubism or the phenotypes exhibited by SH3BP2 cherubism mice under physiological and periodontitis conditions. Unlike bone marrow-derived M-CSF-dependent macrophages (BMMs) carrying the SH3BP2 cherubism mutation, BMMs lacking OGFRL1 or carrying the Syrian mutation showed no difference in TNF-ɑ mRNA induction by LPS or TNF-ɑ compared to WT BMMs. Osteoclast formation induced by RANKL was also comparable. These results suggest that the loss-of-function effects of OGFRL1 in humans differ from those in mice and highlight the fact that mice are not always an ideal model for studying rare craniofacial bone disordersItem Microbe-Dependent Exacerbated Alveolar Bone Destruction in Heterozygous Cherubism Mice(American Society for Bone and Mineral Research, 2020-02-24) Kittaka, Mizuho; Yoshimoto, Tetsuya; Schlosser, Collin; Kajiya, Mikihito; Kurihara, Hidemi; Reichenberger, Ernst J.; Ueki, Yasuyoshi; Biomedical Sciences and Comprehensive Care, School of DentistryCherubism (OMIM#118400) is a craniofacial disorder characterized by destructive jaw expansion. Gain‐of‐function mutations in SH3‐domain binding protein 2 (SH3BP2) are responsible for this rare disorder. We have previously shown that homozygous knock‐in (KI) mice (Sh3bp2 KI/KI) recapitulate human cherubism by developing inflammatory lesions in the jaw. However, it remains unknown why heterozygous KI mice (Sh3bp2 KI/+) do not recapitulate the excessive jawbone destruction in human cherubism, even though all mutations are heterozygous in humans. We hypothesized that Sh3bp2 KI/+ mice need to be challenged for developing exacerbated jawbone destruction and that bacterial stimulation in the oral cavity may be involved in the mechanism. In this study, we applied a ligature‐induced periodontitis model to Sh3bp2 KI/+ mice to induce inflammatory alveolar bone destruction. Ligature placement induced alveolar bone resorption with gingival inflammation. Quantification of alveolar bone volume revealed that Sh3bp2 KI/+ mice developed more severe bone loss (male: 43.0% ± 10.6%, female: 42.6% ± 10.4%) compared with Sh3bp2 +/+ mice (male: 25.8% ± 4.0%, female: 30.9% ± 6.5%). Measurement of bone loss by the cement‐enamel junction–alveolar bone crest distance showed no difference between Sh3bp2 KI/+ and Sh3bp2 +/+ mice. The number of osteoclasts on the alveolar bone surface was higher in male Sh3bp2 KI/+ mice, but not in females, compared with Sh3bp2 +/+ mice. In contrast, inflammatory cytokine levels in gingiva were comparable between Sh3bp2 KI/+ and Sh3bp2 +/+ mice with ligatures. Genetic deletion of the spleen tyrosine kinase in myeloid cells and antibiotic treatment suppressed alveolar bone loss in Sh3bp2 KI/+ mice, suggesting that increased osteoclast differentiation and function mediated by SYK and accumulation of oral bacteria are responsible for the increased alveolar bone loss in Sh3bp2 KI/+ mice with ligature‐induced periodontitis. High amounts of oral bacterial load caused by insufficient oral hygiene could be a trigger for the initiation of jawbone destruction in human cherubism.Item OC_Finder: Osteoclast Segmentation, Counting, and Classification Using Watershed and Deep Learning(Frontiers Media, 2022) Wang, Xiao; Kittaka, Mizuho; He, Yilin; Zhang, Yiwei; Ueki, Yasuyoshi; Kihara, Daisuke; Biomedical Sciences and Comprehensive Care, School of DentistryOsteoclasts are multinucleated cells that exclusively resorb bone matrix proteins and minerals on the bone surface. They differentiate from monocyte/macrophage lineage cells in the presence of osteoclastogenic cytokines such as the receptor activator of nuclear factor-κB ligand (RANKL) and are stained positive for tartrate-resistant acid phosphatase (TRAP). In vitro osteoclast formation assays are commonly used to assess the capacity of osteoclast precursor cells for differentiating into osteoclasts wherein the number of TRAP-positive multinucleated cells is counted as osteoclasts. Osteoclasts are manually identified on cell culture dishes by human eyes, which is a labor-intensive process. Moreover, the manual procedure is not objective and results in lack of reproducibility. To accelerate the process and reduce the workload for counting the number of osteoclasts, we developed OC_Finder, a fully automated system for identifying osteoclasts in microscopic images. OC_Finder consists of cell image segmentation with a watershed algorithm and cell classification using deep learning. OC_Finder detected osteoclasts differentiated from wild-type and Sh3bp2 KI/+ precursor cells at a 99.4% accuracy for segmentation and at a 98.1% accuracy for classification. The number of osteoclasts classified by OC_Finder was at the same accuracy level with manual counting by a human expert. OC_Finder also showed consistent performance on additional datasets collected with different microscopes with different settings by different operators. Together, successful development of OC_Finder suggests that deep learning is a useful tool to perform prompt and accurate unbiased classification and detection of specific cell types in microscopic images.Item Optineurin regulates osteoblastogenesis through STAT1(Elsevier, 2020-05) Mizuno, Noriyoshi; Iwata, Tomoyuki; Ohsawa, Ryosuke; Ouhara, Kazuhisa; Matsuda, Shinji; Kajiya, Mikihito; Matsuda, Yukiko; Kume, Kodai; Tada, Yui; Morino, Hiroyuki; Yoshimoto, Tetsuya; Ueki, Yasuyoshi; Mihara, Keichiro; Sotomaru, Yusuke; Takeda, Katsuhiro; Munenaga, Syuichi; Fujita, Tsuyoshi; Kawaguchi, Hiroyuki; Shiba, Hideki; Kawakami, Hideshi; Kurihara, Hidemi; Biomedical Sciences and Comprehensive Care, School of DentistryA sophisticated and delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts regulates bone metabolism. Optineurin (OPTN) is a gene involved in primary open-angle glaucoma and amyotrophic lateral sclerosis. Although its function has been widely studied in ophthalmology and neurology, recent reports have shown its possible involvement in bone metabolism through negative regulation of osteoclast differentiation. However, little is known about the role of OPTN in osteoblast function. Here, we demonstrated that OPTN controls not only osteoclast but also osteoblast differentiation. Different parameters involved in osteoblastogenesis and osteoclastogenesis were assessed in Optn−/- mice. The results showed that osteoblasts from Optn−/- mice had impaired alkaline phosphatase activity, defective mineralized nodules, and inability to support osteoclast differentiation. Moreover, OPTN could bind to signal transducer and activator of transcription 1 (STAT1) and regulate runt-related transcription factor 2 (RUNX2) nuclear localization by modulating STAT1 levels in osteoblasts. These data suggest that OPTN is involved in bone metabolism not only by regulating osteoclast function but also by regulating osteoblast function by mediating RUNX2 nuclear translocation via STAT1.Item Osteocyte RANKL Drives Bone Resorption in Mouse Ligature‐Induced Periodontitis(Oxford University Press, 2023) Kittaka, Mizuho; Yoshimoto, Tetsuya; Levitan, Marcus E.; Urata, Rina; Choi, Roy B.; Teno, Yayoi; Xie, Yixia; Kitase, Yukiko; Prideaux, Matthew; Dallas, Sarah L.; Robling, Alexander G.; Ueki, Yasuyoshi; Biomedical and Applied Sciences, School of DentistryMouse ligature-induced periodontitis (LIP) has been used to study bone loss in periodontitis. However, the role of osteocytes in LIP remains unclear. Furthermore, there is no consensus on the choice of alveolar bone parameters and time points to evaluate LIP. Here, we investigated the dynamics of changes in osteoclastogenesis and bone volume (BV) loss in LIP over 14 days. Time-course analysis revealed that osteoclast induction peaked on days 3 and 5, followed by the peak of BV loss on day 7. Notably, BV was restored by day 14. The bone formation phase after the bone resorption phase was suggested to be responsible for the recovery of bone loss. Electron microscopy identified bacteria in the osteocyte lacunar space beyond the periodontal ligament (PDL) tissue. We investigated how osteocytes affect bone resorption of LIP and found that mice lacking receptor activator of NF-κB ligand (RANKL), predominantly in osteocytes, protected against bone loss in LIP, whereas recombination activating 1 (RAG1)-deficient mice failed to resist it. These results indicate that T/B cells are dispensable for osteoclast induction in LIP and that RANKL from osteocytes and mature osteoblasts regulates bone resorption by LIP. Remarkably, mice lacking the myeloid differentiation primary response gene 88 (MYD88) did not show protection against LIP-induced bone loss. Instead, osteocytic cells expressed nucleotide-binding oligomerization domain containing 1 (NOD1), and primary osteocytes induced significantly higher Rankl than primary osteoblasts when stimulated with a NOD1 agonist. Taken together, LIP induced both bone resorption and bone formation in a stage-dependent manner, suggesting that the selection of time points is critical for quantifying bone loss in mouse LIP. Pathogenetically, the current study suggests that bacterial activation of osteocytes via NOD1 is involved in the mechanism of osteoclastogenesis in LIP. The NOD1-RANKL axis in osteocytes may be a therapeutic target for bone resorption in periodontitis.Item Osteocytes directly regulate osteolysis via MYD88 signaling in bacterial bone infection(Springer Nature, 2022-11-04) Yoshimoto, Tetsuya; Kittaka, Mizuho; Doan, Andrew Anh Phuong; Urata, Rina; Prideaux, Matthew; Rojas, Roxana E.; Harding, Clifford V.; Boom, W. Henry; Bonewald, Lynda F.; Greenfield, Edward M.; Ueki, Yasuyoshi; Biomedical Sciences and Comprehensive Care, School of DentistryThe impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administration of an MYD88 inhibitor prevents jawbone loss in Pg-driven periodontitis. These findings reveal that osteocytes directly regulate inflammatory osteolysis in bone infection, suggesting that MYD88 and downstream RANKL regulators in osteocytes are therapeutic targets for osteolysis in periodontitis and osteomyelitis.