免疫組織染色法によるヒストンH3リジン9の修飾とメチル化酵素の検出および骨格系細胞分化過程におけるそれらの局在変化

早大学位記番号:新6540早稲田大

Immunohistochemical detection of methylated histone H3 lysine 9 and histone methyltransferases and implication of their roles during skeletal cell differentiation

早大学位記番号:新6540博士(人間科学)早稲田大

Annexin A5 involvement in bone overgrowth at the enthesis

Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5‐deficient (Anxa5–/–) mice, the sizes of bone ridge outgrowths at the entheses of the tibiae and femur were increased after 7 weeks of age. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP‐expressing cells were observed in the fibrocartilage layer in Anxa5–/– mice than in wild‐type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5–/– mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5–/– mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail‐suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5–/– and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of AnxaA5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. AnxaA5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in AnxaA5 KO mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators

Mechanism by which nuclear molecule G9a regulates master genes involved in cell proliferation and differentiation during osteogenesis

研究成果の概要（和文）：本研究は核内分子G9aが細胞系譜特異的マスター遺伝子の機能を調節する仕組みを、骨代謝を担う細胞を用いて明らかにする事を目的とした。その結果、破骨細胞系譜において、G9aノックダウンおよびG9a欠損することで破骨細胞分化マスター遺伝子Nfatc1および増殖関連遺伝子の発現は変動せず、Nfatc1制御下のCathepsin K（Ctsk）の発現が亢進する事、Ctskプロモーター上へのNfatc1の集積が更新する事、G9aとNfatc1が直接結合しない事、が明らかになった。以上から、前駆破骨細胞分化過程にてG9aがNfatc1の機能を間接的に調節する可能性が示唆された。研究成果の概要（英文）：In this study, we aimed to understand how the nuclear molecule G9a influences the function of specific cell lineage master genes in cells related to bone metabolism. The results showed that knocking down G9a and its loss in the osteoclast lineage did not change the expression of the osteoclast differentiation master gene Nfatc1 and proliferation-related genes. However, it did enhance the expression of Nfatc1-regulated Cathepsin K (Ctsk), resulting in renewed Nfatc1 accumulation on the Ctsk promoter. Additionally, it was observed that G9a and Nfatc1 do not bind directly. These findings suggest that G9a may indirectly regulate Nfatc1 function during progenitor osteoclast differentiation.・2023(令和5)年度 科学研究費補助金 基盤研究(C) 研究成果報告書・研究期間 (年度)：2020-04-01 – 2024-03-31・研究分野：分子生物学・研究成果の学術的意義や社会的意義 : 核内分子G9aはH3K9のメチル化を介し、様々な核内タンパク質と相互作用する重要なエピジェネティック修飾因子である。本研究では、G9aがH3K9のメチル化とは異なる仕組みをもって破骨細胞系譜の重要な転写因子NFATc1の機能を調節することによって、破骨細胞形成に抑制的にはたらく新しい可能性が示唆された。研究課題/領域番号 : 20K09897research repor

Search for Conditions to Detect Epigenetic Marks and Nuclear Proteins in Immunostaining of the Testis and Cartilage

The localization of nuclear proteins and modified histone tails changes during cell differentiation at the tissue as well as at the cellular level. Immunostaining in paraffin sections is the most powerful approach available to evaluate protein localization. Since nuclear proteins are sensitive to fixation, immunohistochemical conditions should be optimized in light of the particular antibodies and tissues employed. In this study, we searched for optimal conditions to detect histone modification at histone H3 lysine 9 (H3K9) and H3K9 methyltransferase G9a in the testis and cartilage in paraffin sections. In the testis, antigen retrieval (AR) was indispensable for detecting H3K9me1 and me3, G9a, and nuclear protein proliferating cell nuclear antigen (PCNA). With AR, shorter fixation times yielded better results for the detection of G9a and PCNA. Without AR, H3K9me2 and H3K9ac could be detected at shorter fixation times in primary spermatocytes of the testis. In contrast to the testis, all antibodies tested could detect their epitopes irrespective of AR application in the growth plate cartilage. Thus, conditions for the detection of epigenetic marks and nuclear proteins should be optimized in consideration of fixation time and AR application in different tissues and antibodies.</jats:p

Platelet-derived growth factor-BB regenerates functional periodontal ligament in the tooth replantation

AbstractTooth ankylosis is a pathological condition of periodontal ligament (PDL) restoration after tooth replantation. Platelet-derived growth factor-BB (PDGF-BB) has been proposed as a promising factor for preventing tooth ankylosis. Using rat tooth replantation model, we investigated whether PDGF-BB accelerates the repair of PDL after tooth replantation without ankylosis, and its molecular mechanisms. In PDGF-BB pretreated replanted teeth (PDGF-BB group), ankylosis was markedly reduced and functionally organized PDL collagen fibers were restored; the mechanical strength of the healing PDL was restored to an average of 76% of that in non-replanted normal teeth at 21 days. The numbers of PDGF-Rβ- and BrdU-positive cells in the periodontal tissues of the PDGF-BB group were greater than those of atelocollagen pretreated replanted teeth (AC group). Moreover, in the PDGF-BB group, the periodontal tissues had fewer osteocalcin-positive cells and decreased number of nuclear β-catenin-positive cells compared to those in the AC group. In vitro analyses showed that PDGF-BB increased the proliferation and migration of human periodontal fibroblasts. PDGF-BB downregulated mRNA expressions of RUNX2 and ALP, and inhibited upregulatory effects of Wnt3a on β-catenin, AXIN2, RUNX2, COL1A1, and ALP mRNA expressions. These findings indicate that in tooth replantation, topical PDGF-BB treatment enhances cell proliferation and migration, and inhibits canonical Wnt signaling activation in bone-tooth ankylosis, leading to occlusal loading of the PDL tissues and subsequent functional restoration of the healing PDL. This suggests a possible clinical application of PDGF-BB to reduce ankylosis after tooth replantation and promote proper regeneration of PDL.</jats:p