OP0201 DYNAMIC CHANGES IN O-GLCNACYLATION REGULATE OSTEOCLAST DIFFERENTIATION AND BONE LOSS IN ARTHRITIS

Background:Bone remodeling is a constant process maintained by the balance between osteoclast-triggered bone resorption and osteoblast-mediated bone formation. In inflammatory arthritis, such as rheumatoid arthritis (RA), the pro-inflammatory environment favors osteoclast differentiation and skews the balance towards resorption, leading to progressive bone erosion and bone loss. O-GlcNAcylation is a post-translational modification, which transfers a single N-acetylglucosamine molecule to the serine or threonine of the target protein. The modification is accomplished by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Unlike other glycosylation, O-GlcNAcylation occurs in multiple cellular compartments, including the nucleus. Although O-GlcNAcylation is one of the most common modifications, its role in bone homeostasis is still poorly understood.Objectives:We aimed to investigate the role of O-GlcNAcylation in osteoclastogenesis under pro-inflammatory milieus. We also focused on dissecting the signaling pathways affected by O-GlcNAcylation during osteoclast differentiation.Methods:We examined the levels of O-GlcNAc during in vitro osteoclastogenesis by western blotting. The levels of O-GlcNAc in tissue from RA patients and experimental arthritis were detected by immunofluorescence. Pharmacological inhibition and genetic knockout were used to manipulate O-GlcNAcylaiton during osteoclastogenesis. RNA sequencing was performed to study O-GlcNAc-mediated pathways.Results:We demonstrate the dynamic changes in O-GlcNAcylation during osteoclastogenesis. The elevated O-GlcNAcylation was found in the early differentiation stages, whereas its downregulation was detected in the maturation process. TNFα elaborates the dynamic changes in O-GlcNAcylation, which further intensifies osteoclast differentiation.Targeting OGT by selective inhibitor and genetic knockout restrain O-GlcNAcylation and hinder the expression of the early differentiation marker Nfatc1. Inhibition of OGA, which forces high levels of O-GlcNAcylation throughout the differentiation, reduces the formation of multinucleated mature osteoclasts. Consistent with our in vitro data, suppressing OGT and OGA both ameliorate bone loss in experimental arthritis. We detected a reduced number of TRAP-expressing precursors and mature osteoclasts in the mice subjected to OGT inhibition. While inhibiting OGA only lowers the number of TRAP+F4/80– mature osteoclasts without affecting the number of TRAP+F4/80+ precursors.Transcriptome profiling reveals that O-GlcNAcylation regulates several biological processes. Increased O-GlcNAcylation promotes cytokine signaling and oxidative phosphorylation. The downregulation of O-GlcNAcylation is essential for cytoskeleton organization and cell fusion.Conclusion:We demonstrate that the dynamic changes of O-GlcNAcylation are essential for osteoclast differentiation. These findings reveal the therapeutic potential of targeting O-GlcNAcylation in pathologic bone resorption.Disclosure of Interests:Chih-Wei Chen: None declared, Yi-Nan Li: None declared, Thuong Trinh-Minh: None declared, ZHU Honglin: None declared, Alexandru-Emil Matei: None declared, Xiao Ding: None declared, Cuong Tran Manh: None declared, Xiaohan Xu: None declared, Christoph Liebel: None declared, Ruifang Liang: None declared, Min-Chuan Huang: None declared, Neng-Yu Lin: None declared, Andreas Ramming Speakers bureau: Boehringer Ingelheim, Roche, Janssen, Consultant of: Boehringer Ingelheim, Novartis, Gilead, Pfizer, Grant/research support from: Pfizer, Novartis, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Jörg H.W. Distler Shareholder of: 4D Science, Speakers bureau: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, Grant/research support from: Anamar, Active Biotech, Array Biopharma, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB, Employee of: FibroCure

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Decitabine Inhibits Bone Resorption in Periodontitis by Upregulating Anti-Inflammatory Cytokines and Suppressing Osteoclastogenesis

Biomedicines ◽

10.3390/biomedicines9020199 ◽

2021 ◽

Vol 9 (2) ◽

pp. 199

Author(s):

Urara Tanaka ◽

Shunichi Kajioka ◽

Livia S. Finoti ◽

Daniela B. Palioto ◽

Denis F. Kinane ◽

...

Keyword(s):

Dna Methylation ◽

Bone Resorption ◽

Bone Loss ◽

Inflammatory Cytokines ◽

Osteoclast Differentiation ◽

Tartrate Resistant Acid Phosphatase ◽

Dependent Manner ◽

Bone Homeostasis ◽

Osteoblastic Cell Line ◽

Anti Inflammatory

DNA methylation controls several inflammatory genes affecting bone homeostasis. Hitherto, inhibition of DNA methylation in vivo in the context of periodontitis and osteoclastogenesis has not been attempted. Ligature-induced periodontitis in C57BL/6J mice was induced by placing ligature for five days with Decitabine (5-aza-2′-deoxycytidine) (1 mg/kg/day) or vehicle treatment. We evaluated bone resorption, osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) and mRNA expression of anti-inflammatory molecules using cluster differentiation 14 positive (CD14+) monocytes from human peripheral blood. Our data showed that decitabine inhibited bone loss and osteoclast differentiation experimental periodontitis, and suppressed osteoclast CD14+ human monocytes; and conversely, that it increased bone mineralization in osteoblastic cell line MC3T3-E1 in a concentration-dependent manner. In addition to increasing IL10 (interleukin-10), TGFB (transforming growth factor beta-1) in CD14+ monocytes, decitabine upregulated KLF2 (Krüppel-like factor-2) expression. Overexpression of KLF2 protein enhanced the transcription of IL10 and TGFB. On the contrary, site-directed mutagenesis of KLF2 binding site in IL10 and TFGB abrogated luciferase activity in HEK293T cells. Decitabine reduces bone loss in a mouse model of periodontitis by inhibiting osteoclastogenesis through the upregulation of anti-inflammatory cytokines via KLF2 dependent mechanisms. DNA methyltransferase inhibitors merit further investigation as a possible novel therapy for periodontitis.

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Capsaicin, a TRPV1 Ligand, Suppresses Bone Resorption by Inhibiting the Prostaglandin E Production of Osteoblasts, and Attenuates the Inflammatory Bone Loss Induced by Lipopolysaccharide

ISRN Pharmacology ◽

10.5402/2012/439860 ◽

2012 ◽

Vol 2012 ◽

pp. 1-6 ◽

Cited By ~ 11

Author(s):

Megumi Kobayashi ◽

Kenta Watanabe ◽

Satoshi Yokoyama ◽

Chiho Matsumoto ◽

Michiko Hirata ◽

...

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Transient Receptor Potential ◽

Interleukin 1 ◽

Osteoclast Differentiation ◽

Bone Diseases ◽

Prostaglandin E ◽

Transient Receptor Potential Vanilloid ◽

Cox 2

Capsaicin, a transient receptor potential vanilloid type 1 (TRPV1) ligand, regulates nerve-related pain-sensitive signals, inflammation, and cancer growth. Capsaicin suppresses interleukin-1-induced osteoclast differentiation, but its roles in bone tissues and bone diseases are not known. This study examined the effects of capsaicin on inflammatory bone resorption and prostaglandin E (PGE) production induced by lipopolysaccharide (LPS) in vitro and on bone mass in LPS-treated mice in vivo. Capsaicin suppressed osteoclast formation, bone resorption, and PGE production induced by LPS in vitro. Capsaicin suppressed the expression of cyclooxygenase-2 (COX-2) and membrane-bound PGE synthase-1 (mPGES-1) mRNAs and PGE production induced by LPS in osteoblasts. Capsaicin may suppress PGE production by inhibiting the expression of COX-2 and mPGES-1 in osteoblasts and LPS-induced bone resorption by TRPV1 signals because osteoblasts express TRPV1. LPS treatment markedly induced bone loss in the femur in mice, and capsaicin significantly restored the inflammatory bone loss induced by LPS in mice. TRPV1 ligands like capsaicin may therefore be potentially useful as clinical drugs targeting bone diseases associated with inflammatory bone resorption.

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Pristimerin Inhibits Osteoclast Differentiation and Bone Resorption in vitro and Prevents Ovariectomy-Induced Bone Loss in vivo

Drug Design Development and Therapy ◽

10.2147/dddt.s275128 ◽

2020 ◽

Vol Volume 14 ◽

pp. 4189-4203

Author(s):

Peng Sun ◽

Qichang Yang ◽

Yanben Wang ◽

Jiaxuan Peng ◽

Kangxian Zhao ◽

...

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Osteoclast Differentiation

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Constitutive Activation of β-Catenin in Differentiated Osteoclasts Induces Bone Loss in Mice

Cellular Physiology and Biochemistry ◽

10.1159/000492549 ◽

2018 ◽

Vol 48 (5) ◽

pp. 2091-2102 ◽

Cited By ~ 3

Author(s):

Xin Sui ◽

Shijian Deng ◽

Mengmeng Liu ◽

Linlin Fan ◽

Yunfei Wang ◽

...

Keyword(s):

Bone Loss ◽

Bone Mass ◽

Bone Growth ◽

Osteoclast Differentiation ◽

Marker Genes ◽

Tartrate Resistant Acid Phosphatase ◽

Bone Homeostasis ◽

Micro Computed Tomography ◽

Constitutive Activation

Background/Aims: Activation of the Wnt/β-catenin signalling pathway has been widely investigated in bone biology and shown to promote bone formation. However, its specific effects on osteoclast differentiation have not been fully elucidated. Our study aimed to identify the role of β-catenin in osteoclastogenesis and bone homeostasis. Methods: In the present study, exon 3 in the β-catenin gene (Ctnnb1) allele encoding phosphorylation target serine/threonine residues was flanked by floxP sequences. We generated mice exhibiting conditional β-catenin activation (Ctsk-Cre;Ctnnb1flox(exon3)/+, designated CA-β-catenin) by crossing Ctnnb1flox(exon3)/flox(exon3) mice with osteoclast-specific Ctsk-Cre mice. Bone growth and bone mass were analysed by micro-computed tomography (micro-CT) and histomorphometry. To further examine osteoclast activity, osteoclasts were induced from bone marrow monocytes (BMMs) isolated from CA-β-catenin and Control mice in vitro. Osteoclast differentiation was detected by tartrate-resistant acid phosphatase (TRAP) staining, immunofluorescence staining and reverse transcription-quantitative PCR (RT–qPCR) analysis. Results: Growth retardation and low bone mass were observed in CA-β-catenin mice. Compared to controls, CA-β-catenin mice had significantly reduced trabecular bone numbers under growth plates as well as thinner cortical bones. Moreover, increased TRAP-positive osteoclasts were observed on the surfaces of trabecular bones and cortical bones in the CA-β-catenin mice; consistent results were observed in vitro. In the CA-β-catenin group, excessive numbers of osteoclasts were induced from BMMs, accompanied by the increased expression of osteoclast-associated marker genes. Conclusion: These results indicated that the constitutive activation of β-catenin in osteoclasts promotes osteoclast formation, resulting in bone loss.

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Hedgehog Inhibitors Suppress Osteoclastogenesis in In Vitro Cultures, and Deletion of Smo in Macrophage/Osteoclast Lineage Prevents Age-Related Bone Loss

International Journal of Molecular Sciences ◽

10.3390/ijms21082745 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2745

Author(s):

Yukihiro Kohara ◽

Ryuma Haraguchi ◽

Riko Kitazawa ◽

Yuuki Imai ◽

Sohei Kitazawa

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Signaling Pathway ◽

Early Stage ◽

Osteoclast Differentiation ◽

Osteoclast Formation ◽

Age Related ◽

Hh Signaling

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on Hh functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0–48 h (early stage of osteoclast differentiation) or 48–96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.

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Flemingia macrophyllaExtract Ameliorates Experimental Osteoporosis in Ovariectomized Rats

Evidence-based Complementary and Alternative Medicine ◽

10.1093/ecam/nep179 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 7

Author(s):

Hui-Ya Ho ◽

Jin-Bin Wu ◽

Wen-Chuan Lin

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Bone Marrow Cells ◽

Serum Alkaline Phosphatase ◽

Osteoclast Differentiation ◽

Ethanolic Extract ◽

Ovariectomized Rats ◽

Native Plant ◽

Experimental Osteoporosis

Flemingia macrophylla(Leguminosae), a native plant of Taiwan, is used as folk medicine. Anin vitrostudy showed that a 75% ethanolic extract ofF. macrophylla(FME) inhibited osteoclast differentiation of cultured rat bone marrow cells, and the active component, lespedezaflavanone A (LDF-A), was isolated. It was found that oral administration of FME for 13 weeks suppressed bone loss in ovariectomized rats, an experimental model of osteoporosis. In addition, FME decreased urinary deoxypyridinoline concentrations but did not inhibit serum alkaline phosphatase activities, indicating that it ameliorated bone loss via inhibition of bone resorption. These results suggest that FME may represent a useful remedy for the treatment of bone resorption diseases, such as osteoporosis. In addition, LDF-A could be used as a marker compound to control the quality of FME.

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Transcriptional Modulator Ifrd1 Regulates Osteoclast Differentiation through Enhancing the NF-κB/NFATc1 Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.01075-15 ◽

2016 ◽

Vol 36 (19) ◽

pp. 2451-2463 ◽

Cited By ~ 13

Author(s):

Takashi Iezaki ◽

Kazuya Fukasawa ◽

Gyujin Park ◽

Tetsuhiro Horie ◽

Takashi Kanayama ◽

...

Keyword(s):

Bone Loss ◽

Osteoclast Differentiation ◽

Nuclear Factor Κb ◽

Bone Diseases ◽

Nuclear Factor ◽

Activator Protein 1 ◽

Bone Homeostasis ◽

Activated T Cells

Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Here, we show that the transcriptional coactivator/repressor interferon-related developmental regulator 1 (Ifrd1) is expressed in osteoclast lineages and represents a component of the machinery that regulates bone homeostasis. Ifrd1 expression was transcriptionally regulated in preosteoclasts by receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) through activator protein 1. Global deletion of murineIfrd1increased bone formation and decreased bone resorption, leading to a higher bone mass. Deletion ofIfrd1in osteoclast precursors prevented RANKL-induced bone loss, although no bone loss was observed under normal physiological conditions. RANKL-dependent osteoclastogenesis was impairedin vitroinIfrd1-deleted bone marrow macrophages (BMMs).Ifrd1deficiency increased the acetylation of p65 at residues K122 and K123 via the inhibition of histone deacetylase-dependent deacetylation in BMMs. This repressed the NF-κB-dependent transcription of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), an essential regulator of osteoclastogenesis. These findings suggest that an Ifrd1/NF-κB/NFATc1 axis plays a pivotal role in bone remodelingin vivoand represents a therapeutic target for bone diseases.

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Crif1 Promotes Osteoporosis in Mice after Radiation

10.1101/725408 ◽

2019 ◽

Author(s):

Lixin Xiang ◽

Li Chen ◽

Yang Xiang ◽

Fengjie Li ◽

Xiaomei Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Bone Resorption ◽

Bone Loss ◽

Bone Marrow Cells ◽

Current Knowledge ◽

Osteoclast Differentiation ◽

Protein Docking ◽

Rankl Expression ◽

Marrow Cells

AbstractRadiation induces rapid bone loss and enhances bone resorption and RANKL expression. RANKL provides the crucial signal to induce osteoclast differentiation and plays an important role in bone resorption. However, the mechanisms of radiation-induced osteoporosis are not fully understood. Here, we show that Crif1 expression increases in bone marrow cells after radiation. Conditional Crif1 deletion in bone marrow cells causes decreases in RANKL expression and the RANKL/OPG ratio, and relieves bone loss after radiation in mice. We further demonstrated in vitro that Crif1 promotes RANKL secretion via the cAMP/PKA pathway. Moreover, protein-protein docking screening identified five compounds as Crif1 inhibitors; these compounds dramatically suppressed RANKL secretion and CREB phosphorylation when cells were exposed to forskolin. This study enriches current knowledge of the pathogenesis of osteoporosis and provides insights into potential therapeutic strategies for osteoporosis treatment.

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ELMO1 Regulates RANKL-Stimulated Differentiation and Bone Resorption of Osteoclasts

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.702916 ◽

2021 ◽

Vol 9 ◽

Author(s):

Xinyue Liang ◽

Yafei Hou ◽

Lijuan Han ◽

Shuxiang Yu ◽

Yunyun Zhang ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Bone Resorption ◽

Bone Erosion ◽

Osteoclast Differentiation ◽

Small Gtpases ◽

Bone Diseases ◽

Bone Homeostasis ◽

Nucleotide Exchange

Bone homeostasis is a metabolic balance between the new bone formation by osteoblasts and old bone resorption by osteoclasts. Excessive osteoclastic bone resorption results in low bone mass, which is the major cause of bone diseases such as rheumatoid arthritis. Small GTPases Rac1 is a key regulator of osteoclast differentiation, but its exact mechanism is not fully understood. ELMO and DOCK proteins form complexes that function as guanine nucleotide exchange factors for Rac activation. Here, we report that ELMO1 plays an important role in differentiation and bone resorption of osteoclasts. Osteoclast precursors derived from bone marrow monocytes (BMMs) of Elmo1–/– mice display defective adhesion and migration during differentiation. The cells also have a reduced activation of Rac1, p38, JNK, and AKT in response to RANKL stimulation. Importantly, we show that bone erosion is alleviated in Elmo1–/– mice in a rheumatoid arthritis mouse model. Taken together, our results suggest that ELMO1, as a regulator of Rac1, regulates osteoclast differentiation and bone resorption both in vitro and in vivo.

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The Cytoplasmic Dynein Associated Protein NDE1 Regulates Osteoclastogenesis by Modulating M-CSF and RANKL Signaling Pathways

Cells ◽

10.3390/cells11010013 ◽

2021 ◽

Vol 11 (1) ◽

pp. 13

Author(s):

Bhaba K. Das ◽

Jyoti Gogoi ◽

Aarthi Kannan ◽

Ling Gao ◽

Weirong Xing ◽

...

Keyword(s):

Bone Resorption ◽

Signaling Pathways ◽

Osteoclast Differentiation ◽

Cytoplasmic Dynein ◽

Conditional Knockout ◽

Bone Homeostasis ◽

Cytoskeleton Organization ◽

Osteoclast Precursors

Cytoskeleton organization and lysosome secretion play an essential role in osteoclastogenesis and bone resorption. The cytoplasmic dynein is a molecular motor complex that regulates microtubule dynamics and transportation of cargos/organelles, including lysosomes along the microtubules. LIS1, NDE1, and NDEL1 belong to an evolutionary conserved pathway that regulates dynein functions. Disruption of the cytoplasmic dynein complex and deletion of LIS1 in osteoclast precursors arrest osteoclastogenesis. Nonetheless, the role of NDE1 and NDEL1 in osteoclast biology remains elusive. In this study, we found that knocking-down Nde1 expression by lentiviral transduction of specific shRNAs markedly inhibited osteoclastogenesis in vitro by attenuating the proliferation, survival, and differentiation of osteoclast precursor cells via suppression of signaling pathways downstream of M-CSF and RANKL as well as osteoclast differentiation transcription factor NFATc1. To dissect how NDEL1 regulates osteoclasts and bone homeostasis, we generated Ndel1 conditional knockout mice in myeloid osteoclast precursors (Ndel1ΔlysM) by crossing Ndel1-floxed mice with LysM-Cre mice on C57BL/6J background. The Ndel1ΔlysM mice developed normally. The µCT analysis of distal femurs and in vitro osteoclast differentiation and functional assays in cultures unveiled the similar bone mass in both trabecular and cortical bone compartments as well as intact osteoclastogenesis, cytoskeleton organization, and bone resorption in Ndel1ΔlysM mice and cultures. Therefore, our results reveal a novel role of NDE1 in regulation of osteoclastogenesis and demonstrate that NDEL1 is dispensable for osteoclast differentiation and function.

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