WileyActa Medica Okayama0276-34782025DSOK-0011 Potentially Regulates Circadian Misalignment and Affects Gut Microbiota Composition in Activity-Based Anorexia ModelENHirokiKawaiDepartment of Neuropsychiatry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesNanamiWadaDepartment of Neuropsychiatry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesShinjiSakamotoDepartment of Neuropsychiatry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKenjiMiyazakiSumitomo Pharma Co. LtdTaroKatoSumitomo Pharma Co. LtdYoshihiroHoriuchiSumitomo Pharma Co. LtdHiroshiKiriiDepartment of Animal Applied Microbiology, Okayama University Graduate School of Environmental, Life, Natural Science and TechnologyHoang DuyNguyenDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKenjiHinotsuDepartment of Neuropsychiatry, Okayama University HospitalYoshioOhyaDepartment of Neuropsychiatry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTakahiroAsadaDepartment of Neuropsychiatry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAkiyoshiYokodeDepartment of Neuropsychiatry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesYukoOkahisaDepartment of Neuropsychiatry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHarukoMiyazakiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesManabuTakakiDepartment of Neuropsychiatry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesObjective: Anorexia nervosa (AN) is a metabolic-psychiatric disorder characterized by severe weight loss, hypercortisolemia, and hypothalamic–pituitary–adrenal (HPA) axis activation. In this study, we investigated the effect of inhibiting cortisol regeneration via the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) on the pathophysiology of AN.<br>
Method: Female C57BL/6J mice underwent a 7-day activity-based anorexia (ABA) paradigm, involving 3 h daily feeding and free access to wheels, until 25% body weight loss or experiment completion. Mice were orally treated once daily with a potent 11β-HSD1 inhibitor, DSOK-0011, or vehicle. Body weight, food intake, and activity transitions were recorded; plasma corticosterone and cholesterol levels were measured using a fluorometric assay; gut microbiota were analyzed using 16S rRNA sequencing; and hippocampal glial cells were analyzed using immunohistochemistry.<br>
Results: DSOK-0011-treated mice exhibited a modest but significant increase in postprandial wheel-running activity compared to baseline (4–5 p.m., p = 0.018; 5–6 p.m., p = 0.043), whereas vehicle-treated mice showed higher preprandial activity (9–10 a.m., p = 0.0229). Gut microbiota analysis revealed increased alpha diversity in ABA mice, with a specific enrichment of the Lachnospiraceae family in the DSOK-0011 group. However, DSOK-0011 did not significantly affect body weight, food intake, corticosterone, and lipid levels, or hippocampal glial cell populations.<br>
Conclusion: Inhibition of 11β-HSD1 by DSOK-0011 was associated with microbiota alterations and subtle shifts in activity timing under energy-deficient conditions. These findings suggest that peripheral glucocorticoid metabolism may influence microbial and behavioral responses in the ABA model, although its metabolic impact appears limited in the acute phase.No potential conflict of interest relevant to this article was reported.Springer Science and Business Media LLCActa Medica Okayama0962-88193332024Generation and characterization of cerebellar granule neurons specific knockout mice of Golli-MBP99117ENHarukoMiyazakiDepartment of Molecular Biology and Biochemistry, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of MedicineSakiNishiokaDepartment of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka UniversityTomoyukiYamanakaLaboratory of Structural Neuropathology, Graduate School of Brain Science, Doshisha UniversityManabuAbeDepartment of Animal Model Development, Brain Research Institute, Niigata UniversityYukioImamuraLaboratory of Structural Neuropathology, Graduate School of Brain Science, Doshisha UniversityTomohiroMiyasakaFaculty of Life and Medical Sciences, Doshisha UniversityNobutoKakudaFaculty of Life and Medical Sciences, Doshisha UniversityToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of MedicineTomomiShimogoriLaboratory for Molecular Mechanisms of Brain Development, RIKEN Center for Brain ScienceKazuhiroYamakawaLaboratory for Neurogenetics, RIKEN Center for Brain ScienceMasahitoIkawaDepartment of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka UniversityNobuyukiNukinaLaboratory of Structural Neuropathology, Graduate School of Brain Science, Doshisha UniversityGolli–myelin basic proteins, encoded by the myelin basic protein gene, are widely expressed in neurons and oligodendrocytes in the central nervous system. Further, prior research has shown that Golli–myelin basic protein is necessary for myelination and neuronal maturation during central nervous system development. In this study, we established Golli–myelin basic protein-floxed mice to elucidate the cell-type-specific effects of Golli–myelin basic protein knockout through the generation of conditional knockout mice (Golli–myelin basic proteinsfl/fl; E3CreN), in which Golli–myelin basic proteins were specifically deleted in cerebellar granule neurons, where Golli–myelin basic proteins are expressed abundantly in wild-type mice. To investigate the role of Golli–myelin basic proteins in cerebellar granule neurons, we further performed histopathological analyses of these mice, with results indicating no morphological changes or degeneration of the major cellular components of the cerebellum. Furthermore, behavioral analysis showed that Golli–myelin basic proteinsfl/fl; E3CreN mice were healthy and did not display any abnormal behavior. These results suggest that the loss of Golli–myelin basic proteins in cerebellar granule neurons does not lead to cerebellar perturbations or behavioral abnormalities. This mouse model could therefore be employed to analyze the effect of Golli–myelin basic protein deletion in specific cell types of the central nervous system, such as other neuronal cells and oligodendrocytes, or in lymphocytes of the immune system.No potential conflict of interest relevant to this article was reported.Elsevier BVActa Medica Okayama0168-01022182025Alteration of perineuronal nets and parvalbumin interneurons in prefrontal cortex and hippocampus, and correlation with blood corticosterone in activity-based anorexia model mice104922ENHoang DuyNguyenDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHarukoMiyazakiDepartment of Molecular Biology and Biochemistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesHirokiKawaiDepartment of Neuropsychiatry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesZiyiWangDepartment of Molecular Biology and Biochemistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesShinjiSakamotoDepartment of Neuropsychiatry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesManabuTakakiDepartment of Neuropsychiatry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAnorexia nervosa (AN) is an eating disorder characterized by restricted energy intake, severely underweight status, and frequent hyperactivity. Previous research has shown structural and functional alterations in the medial prefrontal cortex (mPFC) and hippocampus of AN patients. To investigate the pathological mechanism of AN, we analyzed the expression and distribution of parvalbumin (PV) interneurons and perineuronal nets (PNNs), which are implicated in the pathology of neuropsychiatric disorders, in the mPFC and hippocampus dorsal (HPCd) and ventral (HPCv) using an activity-based anorexia (ABA) mouse model. We found that PNN expression and density increased in the mPFC, with minor alterations in the HPCd and HPCv of ABA mice. The expression and distribution of PV neurons were unchanged in the brains of ABA mice, except for a regional decrease in PV-expressing neuron density in the HPCd. Co-localization analysis showed an increased number of PNNs enwrapping PV-negative neurons in the mPFC of ABA mice. Furthermore, the upregulation of PNN expression in the mPFC was positively correlated with elevated blood corticosterone levels, a well-known stress indicator, in ABA mice. Our findings suggest that the increased expression and distribution of PNNs surrounding PV-negative neurons in the mPFC may indicate the pathological mechanisms of AN.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama1661-659625122024Local E-rhBMP-2/β-TCP Application Rescues Osteocyte Dendritic Integrity and Reduces Microstructural Damage in Alveolar Bone Post-Extraction in MRONJ-like Mouse Model6648ENAnh TuanDangDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMitsuakiOnoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesZiyiWangDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesIkueTosaDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesEmilio SatoshiHaraAdvanced Research Center for Oral and Craniofacial Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAkihiroMikaiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesWakanaKitagawaDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTomokoYonezawaDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTakuoKubokiDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesThe pathology of medication-related osteonecrosis of the jaw (MRONJ), often associated with antiresorptive therapy, is still not fully understood. Osteocyte networks are known to play a critical role in maintaining bone homeostasis and repair, but the exact condition of these networks in MRONJ is unknown. On the other hand, the local application of E-coli-derived Recombinant Human Bone Morphogenetic Protein 2/beta-Tricalcium phosphate (E-rhBMP-2/beta-TCP) has been shown to promote bone regeneration and mitigate osteonecrosis in MRONJ-like mouse models, indicating its potential therapeutic application for the treatment of MRONJ. However, the detailed effect of BMP-2 treatment on restoring bone integrity, including its osteocyte network, in an MRONJ condition remains unclear. Therefore, in the present study, by applying a scanning electron microscope (SEM) analysis and a 3D osteocyte network reconstruction workflow on the alveolar bone surrounding the tooth extraction socket of an MRONJ-like mouse model, we examined the effectiveness of BMP-2/beta-TCP therapy on the alleviation of MRONJ-related bone necrosis with a particular focus on the osteocyte network and alveolar bone microstructure (microcrack accumulation). The 3D osteocyte dendritic analysis showed a significant decrease in osteocyte dendritic parameters along with a delay in bone remodeling in the MRONJ group compared to the healthy counterpart. The SEM analysis also revealed a notable increase in the number of microcracks in the alveolar bone surface in the MRONJ group compared to the healthy group. In contrast, all of those parameters were restored in the E-rhBMP-2/beta-TCP-treated group to levels that were almost similar to those in the healthy group. In summary, our study reveals that MRONJ induces osteocyte network degradation and microcrack accumulation, while application of E-rhBMP-2/beta-TCP can restore a compromised osteocyte network and abrogate microcrack accumulation in MRONJ.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama2073-440913102024Exploring the Regulators of Keratinization: Role of BMP-2 in Oral Mucosa807ENXindiMuDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMitsuakiOnoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHa Thi ThuNguyenDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesZiyiWangDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKunZhaoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTaishiKomoriTomokoYonezawaDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTakuoKubokiDepartment of Oral Rehabilitation and Implantology, Okayama University HospitalToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesThe oral mucosa functions as a physico-chemical and immune barrier to external stimuli, and an adequate width of the keratinized mucosa around the teeth or implants is crucial to maintaining them in a healthy and stable condition. In this study, for the first time, bulk RNA-seq analysis was performed to explore the gene expression of laser microdissected epithelium and lamina propria from mice, aiming to investigate the differences between keratinized and non-keratinized oral mucosa. Based on the differentially expressed genes (DEGs) and Gene Ontology (GO) Enrichment Analysis, bone morphogenetic protein 2 (BMP-2) was identified to be a potential regulator of oral mucosal keratinization. Monoculture and epithelial-mesenchymal cell co-culture models in the air-liquid interface (ALI) indicated that BMP-2 has direct and positive effects on epithelial keratinization and proliferation. We further performed bulk RNA-seq of the ALI monoculture stimulated with BMP-2 in an attempt to identify the downstream factors promoting epithelial keratinization and proliferation. Analysis of the DEGs identified, among others, IGF2, ID1, LTBP1, LOX, SERPINE1, IL24, and MMP1 as key factors. In summary, these results revealed the involvement of a well-known growth factor responsible for bone development, BMP-2, in the mechanism of oral mucosal keratinization and proliferation, and pointed out the possible downstream genes involved in this mechanism.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155813532023第55回日本結合組織学会学術大会開催報告174174ENToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityNo potential conflict of interest relevant to this article was reported.Public Library ScienceActa Medica Okayama1932-620317112022Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorptione0277307ENHirofumiFujitaDepartment of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesAoiAndoFaculty of Medicine, Okayama University Medical SchoolYoheiMizusawaFaculty of Medicine, Okayama University Medical SchoolMitsuakiOnoDepartment of Molecular Biology and Biochemistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesTakakoHattoriDepartment of Biochemistry and Molecular Dentistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesMunenoriHabutaDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesSatoshiKubotaDepartment of Biochemistry and Molecular Dentistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesHideyoOhuchiDepartment of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesCysteinyl leukotriene receptor 1 (CysLTR1) is a G protein-coupled receptor for the inflammatory lipid mediators cysteinyl leukotrienes, which are involved in smooth muscle constriction, vascular permeability, and macrophage chemokine release. The Cysltr1 gene encoding CysLTR1 is expressed in the macrophage lineage, including osteoclasts, and the CysLTR1 antagonist Montelukast has been shown to suppress the formation of osteoclasts. However, it currently remains unclear whether CysLTR1 is involved in osteoclast differentiation and bone loss. Therefore, to clarify the role of CysLTR1 in osteoclastogenesis and pathological bone loss, we herein generated CysLTR1 loss-of-function mutant mice by disrupting the cysltr1 gene using the CRISPR-Cas9 system. These mutant mice had a frameshift mutation resulting in a premature stop codon (Cysltr1 KO) or an in-frame mutation causing the deletion of the first extracellular loop (Cysltr1(Delta 105)). Bone marrow macrophages (BMM) from these mutant mice lost the intracellular flux of calcium in response to leukotriene D-4, indicating that these mutants completely lost the activity of CysLTR1 without triggering genetic compensation. However, disruption of the Cysltr1 gene did not suppress the formation of osteoclasts from BMM in vitro. We also demonstrated that the CysLTR1 antagonist Montelukast suppressed the formation of osteoclasts without functional CysLTR1. On the other hand, disruption of the Cysltr1 gene partially suppressed the formation of osteoclasts stimulated by leukotriene D-4 and did not inhibit that by glutathione, functioning as a substrate in the synthesis of cysteinyl leukotrienes. Disruption of the Cysltr1 gene did not affect ovariectomy-induced osteoporosis or lipopolysaccharide-induced bone resorption. Collectively, these results suggest that the CysLT-CysLTR1 axis is dispensable for osteoclast differentiation in vitro and pathological bone loss, while the leukotriene D-4-CysTR1 axis is sufficient to stimulate osteoclast formation. We concluded that the effects of glutathione and Montelukast on osteoclast formation were independent of CysLTR1.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama1422-006722232021Suppression of Bone Necrosis around Tooth Extraction Socket in a MRONJ-like Mouse Model by E-rhBMP-2 Containing Artificial Bone Graft Administration12823ENYukieTanakaDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKyaw ThuAungDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMitsuakiOnoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAkihiroMikaiDepartment of Oral Rehabilitation and Implantology, Okayama University HospitalAnh TuanDangDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesEmilio SatoshiHaraDepartment of Biomaterials, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesIkueTosaDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeiIshibashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAyaOno-KimuraDepartment of Oral Rehabilitation and Implantology, Okayama University HospitalKumikoNawachiDepartment of Oral Rehabilitation and Implantology, Okayama University HospitalTakuoKubokiDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMedication-related osteonecrosis of the jaw (MRONJ) is related to impaired bone healing conditions in the maxillomandibular bone region as a complication of bisphosphonate intake. Although there are several hypotheses for the onset of MRONJ symptoms, one of the possible causes is the inhibition of bone turnover and blood supply leading to bone necrosis. The optimal treatment strategy for MRONJ has not been established either. BMP-2, a member of the TGF-beta superfamily, is well known for regulating bone remodeling and homeostasis prenatally and postnatally. Therefore, the objectives of this study were to evaluate whether cyclophosphamide/zoledronate (CY/ZA) induces necrosis of the bone surrounding the tooth extraction socket, and to examine the therapeutic potential of BMP-2 in combination with the hard osteoinductive biomaterial, beta-tricalcium phosphate (beta-TCP), in the prevention and treatment of alveolar bone loss around the tooth extraction socket in MRONJ-like mice models. First, CY/ZA was intraperitoneally administered for three weeks, and alveolar bone necrosis was evaluated before and after tooth extraction. Next, the effect of BMP-2/beta-TCP was investigated in both MRONJ-like prevention and treatment models. In the prevention model, CY/ZA was continuously administered for four weeks after BMP-2/beta-TCP transplantation. In the treatment model, CY/ZA administration was suspended after transplantation of BMP-2/beta-TCP. The results showed that CY/ZA induced a significant decrease in the number of empty lacunae, a sign of bone necrosis, in the alveolar bone around the tooth extraction socket after tooth extraction. Histological analysis showed a significant decrease in the necrotic alveolar bone around tooth extraction sockets in the BMP-2/beta-TCP transplantation group compared to the non-transplanted control group in both MRONJ-like prevention and treatment models. However, bone mineral density, determined by micro-CT analysis, was significantly higher in the BMP-2/beta-TCP transplanted group than in the control group in the prevention model only. These results clarified that alveolar bone necrosis around tooth extraction sockets can be induced after surgical intervention under CY/ZA administration. In addition, transplantation of BMP-2/beta-TCP reduced the necrotic alveolar bone around the tooth extraction socket. Therefore, a combination of BMP-2/beta-TCP could be an alternative approach for both prevention and treatment of MRONJ-like symptoms.No potential conflict of interest relevant to this article was reported.Frontiers Media SAActa Medica Okayama2296-634X92021Assessment of Possible Contributions of Hyaluronan and Proteoglycan Binding Link Protein 4 to Differential Perineuronal Net Formation at the Calyx of Held730550ENKojiroNojimaDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHarukoMiyazakiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTetsuyaHoriCellular and Molecular Synaptic Function Unit, Okinawa Institute of Science and Technology Graduate UniversityLydiaVargovaDepartment of Neuroscience, Charles University, Second Faculty of MedicineToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesThe calyx of Held is a giant nerve terminal mediating high-frequency excitatory input to principal cells of the medial nucleus of the trapezoid body (MNTB). MNTB principal neurons are enwrapped by densely organized extracellular matrix structures, known as perineuronal nets (PNNs). Emerging evidence indicates the importance of PNNs in synaptic transmission at the calyx of Held. Previously, a unique differential expression of aggrecan and brevican has been reported at this calyceal synapse. However, the role of hyaluronan and proteoglycan binding link proteins (HAPLNs) in PNN formation and synaptic transmission at this synapse remains elusive. This study aimed to assess immunohistochemical evidence for the effect of HAPLN4 on differential PNN formation at the calyx of Held. Genetic deletion of Hapln4 exhibited a clear ectopic shift of brevican localization from the perisynaptic space between the calyx of Held terminals and principal neurons to the neuropil surrounding the whole calyx of Held terminals. In contrast, aggrecan expression showed a consistent localization at the surrounding neuropil, together with HAPLN1 and tenascin-R, in both gene knockout (KO) and wild-type (WT) mice. An in situ proximity ligation assay demonstrated the molecular association of brevican with HAPLN4 in WT and HAPLN1 in gene KO mice. Further elucidation of the roles of HAPLN4 may highlight the developmental and physiological importance of PNN formation in the calyx of Held.</p>No potential conflict of interest relevant to this article was reported.Public Library ScienceActa Medica Okayama1932-62031642021Lack of collagen alpha 6(IV) chain in mice does not cause severe-to-profound hearing loss or cochlear malformation, a distinct phenotype from nonsyndromic hearing loss with COL4A6 missense mutatione0249909ENShaoyingTangDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTomokoYonezawaDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesYukihideMaedaDepartment of Otolaryngology-Head and Neck Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMitsuakiOnoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTakahiroMaebaDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesToruMiyoshiDepartment of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesRyusukeMomotaDepartment of Human Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesYasukoTomonoDivision of Molecular and Cell Biology, Shigei Medical Research InstituteToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesCongenital hearing loss affects 1 in every 1000 births, with genetic mutations contributing to more than 50% of all cases. X-linked nonsyndromic hereditary hearing loss is associated with six loci (DFNX1-6) and five genes. Recently, the missense mutation (c.1771G>A, p.Gly591Ser) in COL4A6, encoding the basement membrane (BM) collagen alpha 6(IV) chain, was shown to be associated with X-linked congenital nonsyndromic hearing loss with cochlear malformation. However, the mechanism by which the COL4A6 mutation impacts hereditary hearing loss has not yet been elucidated. Herein, we investigated Col4a6 knockout (KO) effects on hearing function and cochlear formation in mice. Immunohistochemistry showed that the collagen alpha 6(IV) chain was distributed throughout the mouse cochlea within subepithelial BMs underlying the interdental cells, inner sulcus cells, basilar membrane, outer sulcus cells, root cells, Reissner's membrane, and perivascular BMs in the spiral limbus, spiral ligament, and stria vascularis. However, the click-evoked auditory brainstem response analysis did not show significant changes in the hearing threshold of Col4a6 KO mice compared with wild-type (WT) mice with the same genetic background. In addition, the cochlear structures of Col4a6 KO mice did not exhibit morphological alterations, according to the results of high-resolution micro-computed tomography and histology. Hence, loss of Col4a6 gene expression in mice showed normal click ABR thresholds and normal cochlear formation, which differs from humans with the COL4A6 missense mutation c.1771G>A, p.Gly591Ser. Therefore, the deleterious effects in the auditory system caused by the missense mutation in COL4A6 are likely due to the dominant-negative effects of the alpha 6(IV) chain and/or alpha 5 alpha 6 alpha 5(IV) heterotrimer with an aberrant structure that would not occur in cases with loss of gene expression.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama1422-006721212020Distinct Osteogenic Potentials of BMP-2 and FGF-2 in Extramedullary and Medullary Microenvironments7967ENShujiNoshoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesIkueTosaDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMitsuakiOnoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesEmilio SatoshiHaraDepartment of Biomaterials, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeiIshibashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAkihiroMikaiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesYukieTanakaDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAyaKimura-OnoDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTaishiKomoriDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKenjiMaekawaDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTakuoKubokiDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesBone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2) have been regarded as the major cytokines promoting bone formation, however, several studies have reported unexpected results with failure of bone formation or bone resorption of these growth factors. In this study, BMP-2 and FGF-2 adsorbed into atellocollagen sponges were transplanted into bone defects in the bone marrow-scarce calvaria (extramedullary environment) and bone marrow-abundant femur (medullary environment) for analysis of their in vivo effects not only on osteoblasts, osteoclasts but also on bone marrow cells. The results showed that BMP-2 induced high bone formation in the bone marrow-scarce calvaria, but induced bone resorption in the bone marrow-abundant femurs. On the other hand, FGF-2 showed opposite effects compared to those of BMP-2. Analysis of cellular dynamics revealed numerous osteoblasts and osteoclasts present in the newly-formed bone induced by BMP-2 in calvaria, but none were seen in either control or FGF-2-transplanted groups. On the other hand, in the femur, numerous osteoclasts were observed in the vicinity of the BMP-2 pellet, while a great number of osteoblasts were seen near the FGF-2 pellets or in the control group. Of note, FCM analysis showed that both BMP-2 and FGF-2 administrated in the femur did not significantly affect the hematopoietic cell population, indicating a relatively safe application of the two growth factors. Together, these results indicate that BMP-2 could be suitable for application in extramedullary bone regeneration, whereas FGF-2 could be suitable for application in medullary bone regeneration.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama1422-006721192020BMP-2/beta-TCP Local Delivery for Bone Regeneration in MRONJ-Like Mouse Model7028ENAkihiroMikaiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMitsuakiOnoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesIkueTosaDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesNguyenHa Thi ThuDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesEmilio SatoshiHaraDepartment of Biomaterials, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesShujiNoshoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAyaKimura-OnoDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKumikoNawachiDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTakeshiTakaradaDepartment of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTakuoKubokiDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMedication-related osteonecrosis of the jaw (MRONJ) is a severe pathological condition associated mainly with the long-term administration of bone resorption inhibitors, which are known to induce suppression of osteoclast activity and bone remodeling. Bone Morphogenetic Protein (BMP)-2 is known to be a strong inducer of bone remodeling, by directly regulating osteoblast differentiation and osteoclast activity. This study aimed to evaluate the effects of BMP-2 adsorbed onto beta-tricalcium phosphate (beta-TCP), which is an osteoinductive bioceramic material and allows space retention, on the prevention and treatment of MRONJ in mice. Tooth extraction was performed after 3 weeks of zoledronate (ZA) and cyclophosphamide (CY) administration. For prevention studies, BMP-2/beta-TCP was transplanted immediately after tooth extraction, and the mice were administered ZA and CY for an additional 4 weeks. The results showed that while the tooth extraction socket was mainly filled with a sparse tissue in the control group, bone formation was observed at the apex of the tooth extraction socket and was filled with a dense connective tissue rich in cellular components in the BMP-2/beta-TCP transplanted group. For treatment studies, BMP-2/beta-TCP was transplanted 2 weeks after tooth extraction, and bone formation was followed up for the subsequent 4 weeks under ZA and CY suspension. The results showed that although the tooth extraction socket was mainly filled with soft tissue in the control group, transplantation of BMP-2/beta-TCP could significantly accelerate bone formation, as shown by immunohistochemical analysis for osteopontin, and reduce the bone necrosis in tooth extraction sockets. These data suggest that the combination of BMP-2/beta-TCP could become a suitable therapy for the management of MRONJ.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama0014482738322019Mechanical strain attenuates cytokine-induced ADAMTS9 expression via transient receptor potential vanilloid type 1111556ENTakashiOhtsukiDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityAkiraShinaokaDepartment of Human Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKanaeKumagishi-ShinaokaDepartment of Human Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeiichiAsanoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOmer FarukHatipogluDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityJunkoInagakiDepartment of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKenTakahashiDepartment of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeiichiroNishidaDepartment of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeijiNaruseDepartment of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSatoshiHirohataDepartment of Medical Technology, Graduate School of Health Sciences, Okayama University The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1β and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155812722015基底膜ツールキットとしてのXV/XVV型コラーゲン遺伝子の機能103109ENToshitakaOohashiNo potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6932015Eosinophil Cationic Protein Shows Survival Effect on H9c2 Cardiac Myoblast Cells with Enhanced Phosphorylation of ERK and Akt/GSK-3β under Oxidative Stress145153ENHirokoIshiiShigeshiKamikawaSatoshiHirohataAkifumiMizutaniKojiAbeMasaharuSenoToshitakaOohashiYoshifumiNinomiyaOriginal Article10.18926/AMO/53521Eosinophil cationic protein (ECP) is well known as a cationic protein contained in the basic granules of activated eosinophils. Recent studies have reported that ECP exhibits novel activities on various types of cells, including rat neonatal cardiomyocytes. Here we evaluated the effects of ECP on rat cardiac myoblast H9c2 cells. Our results showed that ECP enhanced the survival of the cells, in part by promoting the ERK and Akt/GSK-3β signaling pathways. ECP attenuated the cytotoxic effects of H2O2 on H9c2 cells as well as the production of reactive oxygen species, the number of apoptotic cells and caspase 3/7 activity in the cells. In conclusion, ECP activated the ERK and Akt/GSK-3β pathways, resulting in anti-oxidative effects on H9c2 cells that attenuated apoptosis.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155812412012ランビエ絞輪周囲のECMによるdiffusion barrier形成と跳躍伝導における役割14ENYokoBekkuYoshifumiNinomiyaToshitakaOohashiNo potential conflict of interest relevant to this article was reported.American College of RheumatologyActa Medica Okayama0004-35915252005ADAMTS-9 is synergistically induced by interleukin-1 and tumor necrosis factor in OUMS-27 chondrosarcoma cells and in human chondrocytes14511460ENKadirDemircanSatoshiHirohataKeiichiroNishidaOmer F.HatipogluToshitakaOohashiTomokoYonezawaSuneel S.ApteYoshifumiNinomiya<p><b>Objective</b><br />
To compare induction of the aggrecanases (ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, and ADAMTS-15) by interleukin-1 (IL-1) and tumor necrosis factor (TNF) in chondrocyte-like OUMS-27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene.</p>
<p><b>Methods</b><br />
OUMS-27 cells were stimulated for different periods of time and with various concentrations of IL-1 and/or TNF. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL-1 and/or TNF. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real-time polymerase chain reaction and Northern blotting. ADAMTS-9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL-1-stimulated OUMS-27 cells was investigated.</p>
<p><b>Results</b><pr>
IL-1 increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL-1 stimulation was greater in chondrocytes than in fibroblasts. The combination of IL-1 and TNF had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS-9 protein was also induced in IL-1-stimulated OUMS-27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 up-regulation in OUMS-27 cells.</p>
<p><b>Conclusion</b><br />
ADAMTS9 is an IL-1- and TNF-inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS-9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS-9 may have a pathologic role in arthritis.</p>No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6322009The 3'-untranslated region of ADAMTS1 regulates its mRNA stability7985ENOmer FarukHatipogluSatoshiHirohataKursat OguzYaykasliMehmet ZeynelCilekKadirDemircanRyokoShinohataTomokoYonezawaToshitakaOohashiShozoKusachiYoshifumiNinomiyaOriginal Article10.18926/AMO/31831<p>ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an inflammatory-induced gene. We have previously reported that ADAMTS1 was strongly but transiently expressed in the infarcted heart. In this study, we investigated whether a 3'-untranslated region (UTR) affects the mRNA stability of this gene. When stimulated with tissue necrosis factor (TNF)-alpha, the expression level of ADAMTS1 mRNA rapidly increased, but the induction of ADAMTS1 mRNA peaked at 6h after stimulation, after which the expression levels of ADAMTS1 mRNA decreased. The 3'-UTR ADAMTS1 mRNA contains multiple adenine and uridine-rich elements, suggesting that the 3'-UTR may regulate gene stability. The addition of actinomycin D, an RNA synthesis inhibitor, demonstrated the decay of induced ADAMTS1 mRNA by TNF-alpha. Furthermore, a region containing multiple AUUUA motifs within the ADAMTS1 3'-UTR destabilized transfected Enhanced Green Fluorescence Protein (EGFP) mRNA expression. These results demonstrated that the ADAMTS1 3'-UTR may regulate the expression of ADAMTS1 mRNA.</p>No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X5612002Human BRAL1 and BCAN genes that belong to the link-module superfamily are tandemly arranged on chromosome 1q21-23.2529ENHiroyukiNomotoToshitakaOohashiSatoshiHirakawaYasuyoshiUekiHiroshiOhtsukiYoshifumiNinomiyaArticle10.18926/AMO/31728<p>We herein determined by fluorescence in situ hybridization the chromosomal localization of 2 human genes, BRAL1 and BCAN, both of which belong to the link-module superfamily, i.e. to the same band of chromosome 1q21-23. Further analysis of the genomic organization of BRAL1 and BCAN revealed that the BRAL1 gene was located 20-kb upstream of the BCAN start site. We isolated a polymorphic dinucleotide (CA) repeat sequence from a genomic clone containing the BCAN gene. High heterozygosity (0.79) makes this polymorphism a useful marker in the study of genetic disorders. Knowledge of the structure of the genes and the marker provides essential information for further analysis of the gene locus at chromosome 1q21-23.</p>
No potential conflict of interest relevant to this article was reported.Acta Medica Okayama1992カサノリ (Acetabularia acetabulum) に存在するCl一輸送性ATPaseに関する研究 反応機構･輸送機構の解明ENNo potential conflict of interest relevant to this article was reported.