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1.
Nat Commun ; 15(1): 5994, 2024 Jul 17.
Article in English | MEDLINE | ID: mdl-39013863

ABSTRACT

Chromatin remodeler ARID1A regulates gene transcription by modulating nucleosome positioning and chromatin accessibility. While ARID1A-mediated stage and lineage-restricted gene regulation during cell fate canalization remains unresolved. Using osteoclastogenesis as a model, we show that ARID1A transcriptionally safeguards the osteoclast (OC) fate canalization during proliferation-differentiation switching at single-cell resolution. Notably, ARID1A is indispensable for the transcriptional apparatus condensates formation with coactivator BRD4/lineage-specifying transcription factor (TF) PU.1 at Nfatc1 super-enhancer during safeguarding the OC fate canalization. Besides, the antagonist function between ARID1A-cBAF and BRD9-ncBAF complex during osteoclastogenesis has been validated with in vitro assay and compound mutant mouse model. Furthermore, the antagonistic function of ARID1A-"accelerator" and BRD9-"brake" both depend on coactivator BRD4-"clutch" during osteoclastogenesis. Overall, these results uncover sophisticated cooperation between chromatin remodeler ARID1A, coactivator, and lineage-specifying TF at super-enhancer of lineage master TF in a condensate manner, and antagonist between distinct BAF complexes in the proper and balanced cell fate canalization.


Subject(s)
Cell Differentiation , Cell Lineage , DNA-Binding Proteins , Osteoclasts , Osteogenesis , Transcription Factors , Animals , Transcription Factors/metabolism , Transcription Factors/genetics , Osteoclasts/metabolism , Osteoclasts/cytology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Mice , Osteogenesis/genetics , Osteogenesis/physiology , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Chromatin Assembly and Disassembly , Gene Expression Regulation , Mice, Inbred C57BL , Cell Proliferation , Single-Cell Analysis , Bromodomain Containing Proteins , Nuclear Proteins
2.
Mol Med Rep ; 30(3)2024 Sep.
Article in English | MEDLINE | ID: mdl-39027997

ABSTRACT

The dental follicle (DF) plays an indispensable role in tooth eruption by regulating bone remodeling through their influence on osteoblast and osteoclast activity. The process of tooth eruption involves a series of intricate regulatory mechanisms and signaling pathways. Disruption of the parathyroid hormone­related protein (PTHrP) in the PTHrP­PTHrP receptor signaling pathway inhibits osteoclast differentiation by DF cells (DFCs), thus resulting in obstructed tooth eruption. Furthermore, parathyroid hormone receptor­1 mutations are linked to primary tooth eruption failure. Additionally, the Wnt/ß­catenin, TGF­ß, bone morphogenetic protein and Hedgehog signaling pathways have crucial roles in DFC involvement in tooth eruption. DFC signal loss or alteration inhibits osteoclast differentiation, affects osteoblast and cementoblast differentiation, and suppresses DFC proliferation, thus resulting in failed tooth eruptions. Abnormal tooth eruption is also associated with a range of systemic syndromes and genetic diseases, predominantly resulting from pathogenic gene mutations. Among these conditions, the following disorders arise due to genetic mutations that disrupt DFCs and impede proper tooth eruption: Cleidocranial dysplasia associated with Runt­related gene 2 gene mutations; osteosclerosis caused by CLCN7 gene mutations; mucopolysaccharidosis type VI resulting from arylsulfatase B gene mutations; enamel renal syndrome due to FAM20A gene mutations; and dentin dysplasia caused by mutations in the VPS4B gene. In addition, regional odontodysplasia and multiple calcific hyperplastic DFs are involved in tooth eruption failure; however, they are not related to gene mutations. The specific mechanism for this effect requires further investigation. To the best of our knowledge, previous reviews have not comprehensively summarized the syndromes associated with DF abnormalities manifesting as abnormal tooth eruption. Therefore, the present review aims to consolidate the current knowledge on DFC signaling pathways implicated in abnormal tooth eruption, and their association with disorders of tooth eruption in genetic diseases and syndromes, thereby providing a valuable reference for future related research.


Subject(s)
Dental Sac , Tooth Eruption , Humans , Dental Sac/metabolism , Mutation , Signal Transduction , Animals , Osteoclasts/metabolism , Osteoclasts/pathology , Receptor, Parathyroid Hormone, Type 1/metabolism , Receptor, Parathyroid Hormone, Type 1/genetics , Cell Differentiation , Parathyroid Hormone-Related Protein/metabolism , Parathyroid Hormone-Related Protein/genetics
3.
FASEB J ; 38(13): e23779, 2024 Jul 15.
Article in English | MEDLINE | ID: mdl-38967255

ABSTRACT

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.


Subject(s)
Bone Resorption , Cell Differentiation , Histone-Lysine N-Methyltransferase , NFATC Transcription Factors , Osteoclasts , Osteogenesis , Animals , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Mice , Osteoclasts/metabolism , Bone Resorption/metabolism , Osteogenesis/physiology , Mice, Knockout , RANK Ligand/metabolism , Mice, Inbred C57BL , Cells, Cultured
4.
Bone Res ; 12(1): 40, 2024 Jul 11.
Article in English | MEDLINE | ID: mdl-38987568

ABSTRACT

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.


Subject(s)
Caspase 8 , Cell Fusion , Osteoclasts , Phosphatidylserines , Phospholipid Transfer Proteins , Caspase 8/metabolism , Caspase 8/genetics , Animals , Osteoclasts/metabolism , Phosphatidylserines/metabolism , Phospholipid Transfer Proteins/metabolism , Phospholipid Transfer Proteins/genetics , Mice , Mice, Inbred C57BL , Bone Resorption/metabolism , Bone Resorption/pathology , Bone Resorption/genetics , Cell Differentiation , RANK Ligand/metabolism
5.
Int J Mol Sci ; 25(13)2024 Jun 30.
Article in English | MEDLINE | ID: mdl-39000355

ABSTRACT

Postmenopausal osteoporosis, characterized by an imbalance between osteoclast-mediated bone resorption and osteoblast-driven bone formation, presents substantial health implications. In this study, we investigated the role of black goat extract (BGE), derived from a domesticated native Korean goat, estrogen-like activity, and osteoprotective effects in vitro. BGE's mineral and fatty acid compositions were analyzed via the ICP-AES method and gas chromatography-mass spectrometry, respectively. In vitro experiments were conducted using MCF-7 breast cancer cells, MC3T3-E1 osteoblasts, and RAW264.7 osteoclasts. BGE exhibits a favorable amount of mineral and fatty acid content. It displayed antimenopausal activity by stimulating MCF-7 cell proliferation and augmenting estrogen-related gene expression (ERα, ERß, and pS2). Moreover, BGE positively impacted osteogenesis and mineralization in MC3T3-E1 cells through Wnt/ß-catenin pathway modulation, leading to heightened expression of Runt-related transcription factor 2, osteoprotegerin, and collagen type 1. Significantly, BGE effectively suppressed osteoclastogenesis by curtailing osteoclast formation and activity in RAW264.7 cells, concurrently downregulating pivotal signaling molecules, including receptor activator of nuclear factor κ B and tumor necrosis factor receptor-associated factor 6. This study offers a shred of preliminary evidence for the prospective use of BGE as an effective postmenopausal osteoporosis treatment.


Subject(s)
Cell Differentiation , Goats , Osteoblasts , Osteoclasts , Osteogenesis , Animals , Mice , RAW 264.7 Cells , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteogenesis/drug effects , Cell Differentiation/drug effects , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteoclasts/cytology , Humans , Estrogens/pharmacology , Cell Proliferation/drug effects , Wnt Signaling Pathway/drug effects , MCF-7 Cells , Tissue Extracts/pharmacology
6.
Commun Biol ; 7(1): 892, 2024 Jul 23.
Article in English | MEDLINE | ID: mdl-39039245

ABSTRACT

Bone is a highly dynamic tissue undergoing continuous formation and resorption. Here, we investigated differential but complementary roles of hypoxia-inducible factor (HIF)-1α and HIF-2α in regulating bone remodeling. Using RNA-seq analysis, we identified that specific genes involved in regulating osteoblast differentiation were similarly but slightly differently governed by HIF-1α and HIF-2α. We found that increased HIF-1α expression inhibited osteoblast differentiation via inhibiting RUNX2 function by upregulation of Twist2, confirmed using Hif1a conditional knockout (KO) mouse. Ectopic expression of HIF-1α via adenovirus transduction resulted in the increased expression and activity of RANKL, while knockdown of Hif1a expression via siRNA or osteoblast-specific depletion of Hif1a in conditional KO mice had no discernible effect on osteoblast-mediated osteoclast activation. The unexpected outcome was elucidated by the upregulation of HIF-2α upon Hif1a overexpression, providing evidence that Hif2a is a transcriptional target of HIF-1α in regulating RANKL expression, verified through an experiment of HIF-2α knockdown after HIF-1α overexpression. The above results were validated in an ovariectomized- and aging-induced osteoporosis model using Hif1a conditional KO mice. Our findings conclude that HIF-1α plays an important role in regulating bone homeostasis by controlling osteoblast differentiation, and in influencing osteoclast formation through the regulation of RANKL secretion via HIF-2α modulation.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Homeostasis , Hypoxia-Inducible Factor 1, alpha Subunit , Mice, Knockout , Osteoblasts , Animals , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Mice , Osteoblasts/metabolism , Female , Bone and Bones/metabolism , Cell Differentiation , Osteoclasts/metabolism , Osteogenesis/genetics , Mice, Inbred C57BL , Osteoporosis/genetics , Osteoporosis/metabolism
7.
PeerJ ; 12: e17722, 2024.
Article in English | MEDLINE | ID: mdl-39006031

ABSTRACT

Background: Osteoporosis is a disease associated with bone resorption, characterized primarily by the excessive activation of osteoclasts. Ginkgetin is a compound purified from natural ginkgo leaves which has various biological properties, including anti-inflammation, antioxidant, and anti-tumor effects. This study investigated the bone-protective effects of ginkgetin in ovariectomized (OVX) mice and explored their potential signaling pathway in inhibiting osteoclastogenesis in a mouse model of osteoporosis. Methods: Biochemical assays were performed to assess the levels of Ca, ALP, and P in the blood. Micro CT scanning was used to evaluate the impact of ginkgetin on bone loss in mice. RT-PCR was employed to detect the expression of osteoclast-related genes (ctsk, c-fos, trap) in their femoral tissue. Hematoxylin and eosin (H&E) staining was utilized to assess the histopathological changes in femoral tissue due to ginkgetin. The TRAP staining was used to evaluate the impact of ginkgetin osteoclast generation in vivo. Western blot analysis was conducted to investigate the effect of ginkgetin on the expression of p-NF-κB p65 and IκBα proteins in mice. Results: Our findings indicate that ginkgetin may increase the serum levels of ALP and P, while decreasing the serum level of Ca in OVX mice. H&E staining and micro CT scanning results suggest that ginkgetin can inhibit bone loss in OVX mice. The TRAP staining results showed ginkgetin suppresses the generation of osteoclasts in OVX mice. RT-PCR results demonstrate that ginkgetin downregulate the expression of osteoclast-related genes (ctsk, c-fos, trap) in the femoral tissue of mice, and this effect is dose-dependent. Western blot analysis results reveal that ginkgetin can inhibit the expression of p-NF-κB p65 and IκBα proteins in mice. Conclusion: Ginkgetin can impact osteoclast formation and activation in OVX mice by inhibiting the NF-κB/IκBα signaling pathway, thereby attenuating bone loss in mice.


Subject(s)
Biflavonoids , NF-kappa B , Osteoclasts , Signal Transduction , Animals , Biflavonoids/pharmacology , Biflavonoids/therapeutic use , Signal Transduction/drug effects , Mice , NF-kappa B/metabolism , Female , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteoporosis/drug therapy , Osteoporosis/metabolism , Osteoporosis/pathology , Ovariectomy , Disease Models, Animal , Bone Resorption/drug therapy , Bone Resorption/metabolism , Bone Resorption/prevention & control , Bone Resorption/pathology , X-Ray Microtomography , NF-KappaB Inhibitor alpha/metabolism , Mice, Inbred C57BL
8.
Shanghai Kou Qiang Yi Xue ; 33(2): 130-134, 2024 Apr.
Article in Chinese | MEDLINE | ID: mdl-39005087

ABSTRACT

PURPOSE: To investigate the therapeutic effect of atorvastatin on alveolar bone defect model in rats, and to observe the effect of atorvastatin on Wnt/ß-catenin. METHODS: Thirty rats were randomly divided into normal group (group N), model group (group M) and atorvastatin administration group (group ATV). Except group N, bone defects were made in other rats' alveolar bone to construct alveolar bone defect model. After successful modeling, 20 mg/kg atorvastatin suspension was administered by gavage in group ATV, and the same amount of sodium carboxymethyl cellulose solution was administered by gavage in group N and group M for twenty-one days. After the last administration, tail vein blood was collected to detect the concentrations of serum osteoprotegerin (OPG), alkaline phosphatase (ALP) and osteocalcin (BPG). H-E staining was used to observe the pathological changes of maxillary defect area, and lane Sandhu score was performed. Tartrate resistant acid phosphatase(TRAP) staining was used to detect the number of osteoclasts in the defect area. Real time fluorescence quantitative PCR(RT-qPCR) and Western blot(WB) were used to detect Wnt, ß-catenin and Runx2 mRNA protein expression. Statistical analysis was performed with SPSS 23.0 software package. RESULTS: Compared with group N, the concentrations of OPG, ALP, BGP and Lane Sandhu score in group M decreased, and the number of osteoclasts increased. Compared with group M, the concentrations of OPG, ALP and BGP and lane Sandhu score in group ATV increased, and the number of osteoclasts decreased. After H-E staining, the amount of bone formation in maxillary defect area in group N was more,there was fewer bone tissues in the defect area in group M, the amount of bone tissues in the defect area increased in group ATV. Compared with group N, Wnt, ß-catenin and Runx2 mRNA protein decreased. Compared with group M, Wnt, ß-catenin and Runx2 mRNA protein expression increased. CONCLUSIONS: Atorvastatin can promote the healing of alveolar bone defect and accelerate bone reconstruction in rat models. This effect may be related to the activation of Wnt/ß-catenin signaling pathway.


Subject(s)
Alkaline Phosphatase , Atorvastatin , Osteocalcin , Osteoprotegerin , Wnt Signaling Pathway , beta Catenin , Animals , Atorvastatin/pharmacology , Wnt Signaling Pathway/drug effects , Rats , Osteoprotegerin/metabolism , Osteoprotegerin/genetics , beta Catenin/metabolism , beta Catenin/genetics , Osteocalcin/metabolism , Osteocalcin/genetics , Osteocalcin/blood , Alkaline Phosphatase/metabolism , Alkaline Phosphatase/blood , Osteoclasts/drug effects , Osteoclasts/metabolism , Core Binding Factor Alpha 1 Subunit/metabolism , Core Binding Factor Alpha 1 Subunit/genetics , Alveolar Process/drug effects , Alveolar Process/metabolism
9.
Cells ; 13(13)2024 Jun 24.
Article in English | MEDLINE | ID: mdl-38994943

ABSTRACT

Gingival fibroblasts (GFs) can differentiate into osteoblast-like cells and induce osteoclast precursors to differentiate into osteoclasts. As it is unclear whether these two processes influence each other, we investigated how osteogenic differentiation of GFs affects their osteoclast-inducing capacity. To establish step-wise mineralization, GFs were cultured in four groups for 3 weeks, without or with osteogenic medium for the final 1, 2, or all 3 weeks. The mineralization was assessed by ALP activity, calcium concentration, scanning electron microscopy (SEM), Alizarin Red staining, and quantitative PCR (qPCR). To induce osteoclast differentiation, these cultures were then co-cultured for a further 3 weeks with peripheral blood mononuclear cells (PBMCs) containing osteoclast precursors. Osteoclast formation was assessed at different timepoints with qPCR, enzyme-linked immunosorbent assay (ELISA), TRAcP activity, and staining. ALP activity and calcium concentration increased significantly over time. As confirmed with the Alizarin Red staining, SEM images showed that the mineralization process occurred over time. Osteoclast numbers decreased in the GF cultures that had undergone osteogenesis. TNF-α secretion, a costimulatory molecule for osteoclast differentiation, was highest in the control group. GFs can differentiate into osteoblast-like cells and their degree of differentiation reduces their osteoclast-inducing capacity, indicating that, with appropriate stimulation, GFs could be used in regenerative periodontal treatments.


Subject(s)
Cell Differentiation , Fibroblasts , Gingiva , Osteoclasts , Osteogenesis , Humans , Osteoclasts/metabolism , Osteoclasts/cytology , Gingiva/cytology , Fibroblasts/metabolism , Fibroblasts/cytology , Cells, Cultured , Calcium/metabolism , Tumor Necrosis Factor-alpha/metabolism , Coculture Techniques , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism
10.
Cell Mol Biol Lett ; 29(1): 100, 2024 Jul 08.
Article in English | MEDLINE | ID: mdl-38977961

ABSTRACT

OBJECTIVE: Osteoporosis is a global health issue characterized by decreased bone mass and microstructural degradation, leading to an increased risk of fractures. This study aims to explore the molecular mechanism by which P2X7 receptors influence osteoclast formation and bone resorption through the PI3K-Akt-GSK3ß signaling pathway. METHODS: An osteoporosis mouse model was generated through ovariectomy (OVX) in normal C57BL/6 and P2X7f/f; LysM-cre mice. Osteoclasts were isolated for transcriptomic analysis, and differentially expressed genes were selected for functional enrichment analysis. Metabolite analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and multivariate statistical analysis and pattern recognition were used to identify differential lipid metabolism markers and their distribution. Bioinformatics analyses were conducted using the Encyclopedia of Genes and Genomes database and the MetaboAnalyst database to assess potential biomarkers and create a metabolic pathway map. Osteoclast precursor cells were used for in vitro cell experiments, evaluating cell viability and proliferation using the Cell Counting Kit 8 (CCK-8) assay. Osteoclast precursor cells were induced to differentiate into osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-beta ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) staining was performed to compare differentiation morphology, size, and quantity between different groups. Western blot analysis was used to assess the expression of differentiation markers, fusion gene markers, and bone resorption ability markers in osteoclasts. Immunofluorescence staining was employed to examine the spatial distribution and quantity of osteoclast cell skeletons, P2X7 protein, and cell nuclei, while pit assay was used to evaluate osteoclast bone resorption ability. Finally, in vivo animal experiments, including micro computed tomography (micro-CT), hematoxylin and eosin (HE) staining, TRAP staining, and immunohistochemistry, were conducted to observe bone tissue morphology, osteoclast differentiation, and the phosphorylation level of the PI3K-Akt-GSK3ß signaling pathway. RESULTS: Transcriptomic and metabolomic data collectively reveal that the P2X7 receptor can impact the pathogenesis of osteoporosis through the PI3K-Akt-GSK3ß signaling pathway. Subsequent in vitro experiments showed that cells in the Sh-P2X7 + Recilisib group exhibited increased proliferative activity (1.15 versus 0.59), higher absorbance levels (0.68 versus 0.34), and a significant increase in resorption pit area (13.94 versus 3.50). Expression levels of osteoclast differentiation-related proteins MMP-9, CK, and NFATc1 were markedly elevated (MMP-9: 1.72 versus 0.96; CK: 2.54 versus 0.95; NFATc1: 3.05 versus 0.95), along with increased fluorescent intensity of F-actin rings. In contrast, the OE-P2X7 + LY294002 group showed decreased proliferative activity (0.64 versus 1.29), reduced absorbance (0.34 versus 0.82), and a significant decrease in resorption pit area (5.01 versus 14.96), accompanied by weakened expression of MMP-9, CK, and NFATc1 (MMP-9: 1.14 versus 1.79; CK: 1.26 versus 2.75; NFATc1: 1.17 versus 2.90) and decreased F-actin fluorescent intensity. Furthermore, in vivo animal experiments demonstrated that compared with the wild type (WT) + Sham group, mice in the WT + OVX group exhibited significantly increased levels of CTX and NTX in serum (CTX: 587.17 versus 129.33; NTX: 386.00 versus 98.83), a notable decrease in calcium deposition (19.67 versus 53.83), significant reduction in bone density, increased trabecular separation, and lowered bone mineral density (BMD). When compared with the KO + OVX group, mice in the KO + OVX + recilisib group showed a substantial increase in CTX and NTX levels in serum (CTX: 503.50 versus 209.83; NTX: 339.83 versus 127.00), further reduction in calcium deposition (29.67 versus 45.33), as well as decreased bone density, increased trabecular separation, and reduced BMD. CONCLUSION: P2X7 receptors positively regulate osteoclast formation and bone resorption by activating the PI3K-Akt-GSK3ß signaling pathway.


Subject(s)
Bone Resorption , Cell Differentiation , Glycogen Synthase Kinase 3 beta , Mice, Inbred C57BL , Osteoclasts , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Receptors, Purinergic P2X7 , Signal Transduction , Animals , Osteoclasts/metabolism , Bone Resorption/metabolism , Bone Resorption/genetics , Bone Resorption/pathology , Cell Differentiation/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Glycogen Synthase Kinase 3 beta/genetics , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/genetics , Receptors, Purinergic P2X7/metabolism , Receptors, Purinergic P2X7/genetics , Female , Osteoporosis/metabolism , Osteoporosis/genetics , Osteoporosis/pathology , RANK Ligand/metabolism , RANK Ligand/genetics
11.
Biochem Biophys Res Commun ; 727: 150317, 2024 Oct 01.
Article in English | MEDLINE | ID: mdl-38959733

ABSTRACT

Abnormalities in osteoclastic generation or activity disrupt bone homeostasis and are highly involved in many pathologic bone-related diseases, including rheumatoid arthritis, osteopetrosis, and osteoporosis. Control of osteoclast-mediated bone resorption is crucial for treating these bone diseases. However, the mechanisms of control of osteoclastogenesis are incompletely understood. In this study, we identified that inosine 5'-monophosphate dehydrogenase type II (Impdh2) positively regulates bone resorption. By histomorphometric analysis, Impdh2 deletion in mouse myeloid lineage cells (Impdh2LysM-/- mice) showed a high bone mass due to the reduced osteoclast number. qPCR and western blotting results demonstrated that the expression of osteoclast marker genes, including Nfatc1, Ctsk, Calcr, Acp5, Dcstamp, and Atp6v0d2, was significantly decreased in the Impdh2LysM-/- mice. Furthermore, the Impdh inhibitor MPA treatment inhibited osteoclast differentiation and induced Impdh2-cytoophidia formation. The ability of osteoclast differentiation was recovered after MPA deprivation. Interestingly, genome-wide analysis revealed that the osteoclastic mitochondrial biogenesis and functions, such as oxidative phosphorylation, were impaired in the Impdh2LysM-/- mice. Moreover, the deletion of Impdh2 alleviated ovariectomy-induced bone loss. In conclusion, our findings revealed a previously unrecognized function of Impdh2, suggesting that Impdh2-mediated mechanisms represent therapeutic targets for osteolytic diseases.


Subject(s)
IMP Dehydrogenase , Mitochondria , Osteoclasts , Osteogenesis , Osteoporosis , Ovariectomy , Oxidative Phosphorylation , Animals , Osteoporosis/metabolism , Osteoporosis/etiology , Osteoporosis/genetics , Osteoporosis/pathology , Mice , Female , Osteoclasts/metabolism , Osteoclasts/pathology , Mitochondria/metabolism , Mitochondria/pathology , IMP Dehydrogenase/metabolism , IMP Dehydrogenase/genetics , IMP Dehydrogenase/deficiency , Mice, Knockout , Mice, Inbred C57BL , Cell Differentiation , Bone Resorption/metabolism , Bone Resorption/genetics , Bone Resorption/pathology , Bone Resorption/etiology
12.
Bioorg Chem ; 150: 107603, 2024 Sep.
Article in English | MEDLINE | ID: mdl-38968905

ABSTRACT

Inhibition of LSD1 was proposed as promising and attractive therapies for treating osteoporosis. Here, we synthesized a series of novel TCP-(MP)-Caffeic acid analogs as potential LSD1 inhibitors to assess their inhibitory effects on osteoclastogenesis by using TRAP-staining assay and try to explore the preliminary SAR. Among them, TCP-MP-CA (11a) demonstrated osteoclastic bone loss both in vitro and in vivo, showing a significant improvement in the in vivo effects compared to the LSD1 inhibitor GSK-LSD1. Additionally, we elucidated a mechanism that 11a and its precursor that 11e directly bind to LSD1/CoREST complex through FAD to inhibit LSD1 demethylation activity and influence its downstream IκB/NF-κB signaling pathway, and thus regulate osteoclastic bone loss. These findings suggested 11a or 11e as potential novel candidates for treating osteoclastic bone loss, and a concept for further development of TCP-(MP)-Caffeic acid analogs for therapeutic use in osteoporosis clinics.


Subject(s)
Caffeic Acids , Osteoclasts , Osteoclasts/drug effects , Osteoclasts/metabolism , Caffeic Acids/pharmacology , Caffeic Acids/chemistry , Caffeic Acids/chemical synthesis , Animals , Structure-Activity Relationship , Mice , Molecular Structure , Dose-Response Relationship, Drug , Drug Discovery , Humans , Osteoporosis/drug therapy , Bone Resorption/drug therapy , RAW 264.7 Cells , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis
13.
Sci Adv ; 10(28): eadl4913, 2024 Jul 12.
Article in English | MEDLINE | ID: mdl-38985878

ABSTRACT

The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored mechanisms of silica-induced pulmonary fibrosis in human lung samples collected from patients with occupational exposure to silica and in a longitudinal mouse model of silicosis using multiple modalities including whole-lung single-cell RNA sequencing and histological, biochemical, and physiologic assessments. In addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor κΒ ligand (RANKL) in pulmonary lymphocytes, and alveolar type II cells. Anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated pulmonary fibrosis. We conclude that silica induces differentiation of pulmonary osteoclast-like cells leading to progressive lung injury, likely due to sustained elaboration of bone-resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.


Subject(s)
Cell Differentiation , Osteoclasts , Pulmonary Fibrosis , Silicon Dioxide , Silicosis , Silicon Dioxide/toxicity , Animals , Humans , Osteoclasts/metabolism , Osteoclasts/drug effects , Osteoclasts/pathology , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/metabolism , Mice , Silicosis/pathology , Silicosis/metabolism , Silicosis/etiology , Cell Differentiation/drug effects , RANK Ligand/metabolism , Disease Models, Animal , Male , Lung/pathology , Lung/metabolism , Lung/drug effects , Macrophages, Alveolar/metabolism , Macrophages, Alveolar/pathology , Macrophages, Alveolar/drug effects , Female
14.
J Exp Clin Cancer Res ; 43(1): 183, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38951916

ABSTRACT

BACKGROUND: Leukocyte Ig-like receptor B family 4 (LILRB4) as an immune checkpoint on myeloid cells is a potential target for tumor therapy. Extensive osteolytic bone lesion is the most characteristic feature of multiple myeloma. It is unclear whether ectopic LILRB4 on multiple myeloma regulates bone lesion. METHODS: The conditioned medium (CM) from LILRB4-WT and -KO cells was used to analyze the effects of LILRB4 on osteoclasts and osteoblasts. Xenograft, syngeneic and patient derived xenograft models were constructed, and micro-CT, H&E staining were used to observe the bone lesion. RNA-seq, cytokine array, qPCR, the activity of luciferase, Co-IP and western blotting were used to clarify the mechanism by which LILRB4 mediated bone damage in multiple myeloma. RESULTS: We comprehensively analyzed the expression of LILRB4 in various tumor tissue arrays, and found that LILRB4 was highly expressed in multiple myeloma samples. The patient's imaging data showed that the higher the expression level of LILRB4, the more serious the bone lesion in patients with multiple myeloma. The conditioned medium from LILRB4-WT not -KO cells could significantly promote the differentiation and maturation of osteoclasts. Xenograft, syngeneic and patient derived xenograft models furtherly confirmed that LILRB4 could mediate bone lesion of multiple myeloma. Next, cytokine array was performed to identify the differentially expressed cytokines, and RELT was identified and regulated by LILRB4. The overexpression or exogenous RELT could regenerate the bone damage in LILRB4-KO cells in vitro and in vivo. The deletion of LILRB4, anti-LILRB4 alone or in combination with bortezomib could significantly delay the progression of bone lesion of multiple myeloma. CONCLUSIONS: Our findings indicated that LILRB4 promoted the bone lesion by promoting the differentiation and mature of osteoclasts through secreting RELT, and blocking LILRB4 singling pathway could inhibit the bone lesion.


Subject(s)
Multiple Myeloma , Receptors, Immunologic , Signal Transduction , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Multiple Myeloma/genetics , Humans , Mice , Animals , Receptors, Immunologic/metabolism , Receptors, Immunologic/genetics , NF-kappa B/metabolism , Membrane Glycoproteins/metabolism , Membrane Glycoproteins/genetics , Cell Line, Tumor , Osteoclasts/metabolism , Xenograft Model Antitumor Assays
15.
Chem Biol Drug Des ; 104(1): e14574, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38958121

ABSTRACT

To develop novel bovine lactoferrin (bLF) peptides targeting bLF-tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6) binding sites, we identified two peptides that could target bLF-TRAF6 binding sites using structural analysis. Moreover, another peptide that could bind to the TRAF6 dimerization area was selected from the bLF sequence. The effects of each peptide on cytokine expression in lipopolysaccharide (LPS)-stimulated osteoblasts (ST2) and on osteoclastogenesis were examined using an LPS-treated co-culture of primary bone marrow cells (BMCs) with ST2 cells and a single culture of osteoclast precursor cells (RAW-D) treated with soluble receptor activator of NF-κB ligand. Finally, the effectiveness of these peptides against LPS-induced alveolar bone destruction was assessed. Two of the three peptides significantly suppressed LPS-induced TNF-α and interleukin-1ß expression in ST2 cells. Additionally, these peptides inhibited and reversed LPS-induced receptor activator of NF-κB ligand (RANKL) upregulation and osteoprotegerin (OPG) downregulation, respectively. Furthermore, both peptides significantly reduced LPS-induced osteoclastogenesis in the BMC-ST2 co-culture and RANKL-induced osteoclastogenesis in RAW-D cells. In vivo, topical application of these peptides significantly reduced the osteoclast number by downregulating RANKL and upregulating OPG in the periodontal ligament. It is indicated that the novel bLF peptides can be used to treat periodontitis-associated bone destruction.


Subject(s)
Lactoferrin , Lipopolysaccharides , Osteoclasts , Peptides , Animals , Lactoferrin/pharmacology , Lactoferrin/chemistry , Lactoferrin/metabolism , Lipopolysaccharides/pharmacology , Rats , Peptides/pharmacology , Peptides/chemistry , Osteoclasts/drug effects , Osteoclasts/metabolism , RANK Ligand/metabolism , Male , Alveolar Bone Loss/drug therapy , Alveolar Bone Loss/metabolism , Alveolar Bone Loss/pathology , Cattle , Mice , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteoblasts/cytology , Rats, Sprague-Dawley , Osteogenesis/drug effects , Tumor Necrosis Factor-alpha/metabolism , Binding Sites , Coculture Techniques , Osteoprotegerin/metabolism , Disease Models, Animal
16.
Cell Biol Toxicol ; 40(1): 52, 2024 Jul 05.
Article in English | MEDLINE | ID: mdl-38967699

ABSTRACT

Diabetic osteoporosis (DO) presents significant clinical challenges. This study aimed to investigate the potential of magnetic nanoparticle-enhanced extracellular vesicles (GMNPE-EVs) derived from bone marrow mesenchymal stem cells (BMSCs) to deliver miR-15b-5p, thereby targeting and downregulating glial fibrillary acidic protein (GFAP) expression in rat DO models. Data was sourced from DO-related RNA-seq datasets combined with GEO and GeneCards databases. Rat primary BMSCs, bone marrow-derived macrophages (BMMs), and osteoclasts were isolated and cultured. EVs were separated, and GMNPE targeting EVs were synthesized. Bioinformatic analysis revealed a high GFAP expression in DO-related RNA-seq and GSE26168 datasets for disease models. Experimental results confirmed elevated GFAP in rat DO bone tissues, promoting osteoclast differentiation. miR-15b-5p was identified as a GFAP inhibitor, but was significantly downregulated in DO and enriched in BMSC-derived EVs. In vitro experiments showed that GMNPE-EVs could transfer miR-15b-5p to osteoclasts, downregulating GFAP and inhibiting osteoclast differentiation. In vivo tests confirmed the therapeutic potential of this approach in alleviating rat DO. Collectively, GMNPE-EVs can effectively deliver miR-15b-5p to osteoclasts, downregulating GFAP expression, and hence, offering a therapeutic strategy for rat DO.


Subject(s)
Extracellular Vesicles , Glial Fibrillary Acidic Protein , Mesenchymal Stem Cells , MicroRNAs , Osteoclasts , Osteoporosis , Rats, Sprague-Dawley , Animals , MicroRNAs/genetics , MicroRNAs/metabolism , Mesenchymal Stem Cells/metabolism , Extracellular Vesicles/metabolism , Extracellular Vesicles/genetics , Osteoporosis/metabolism , Osteoporosis/genetics , Glial Fibrillary Acidic Protein/metabolism , Glial Fibrillary Acidic Protein/genetics , Rats , Osteoclasts/metabolism , Male , Cell Differentiation , Magnetite Nanoparticles , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/genetics , Diabetes Complications/metabolism , Diabetes Complications/genetics
17.
J Pharm Biomed Anal ; 248: 116273, 2024 Sep 15.
Article in English | MEDLINE | ID: mdl-38878451

ABSTRACT

Glucocorticoid-induced osteoporosis (GIOP) represents the most prevalent form of secondary osteoporosis. Aucubin (AU), a principal active component found in traditional herbal medicines such as Eucommia ulmoides, has been demonstrated to enhance osteoblast differentiation. Nonetheless, the precise therapeutic effects of AU on GIOP and the complex underlying regulatory mechanisms warrant further investigation. We first established a GIOP model in female mice and then assessed the therapeutic effects of AU using micro-CT analysis, biomechanical testing, measurements of serum calcium (Ca) and phosphorus (P) levels, and histological analyses using Hematoxylin and Eosin (HE) and Masson staining. Subsequently, non-targeted metabolomics was employed in order to study the effects of AU on serum metabolites in GIOP mice. The levels of the factors related to these metabolites were quantified using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blot analyses. Finally, the effects of AU on osteoblastic and osteoclastic differentiation were examined. We found that AU significantly ameliorated bone microarchitecture and strength in GIOP mice. It mitigated pathological damages such as impairment of trabecular bone structure and reduction in collagen fibers, while concurrently elevating serum levels of Ca and P. Non-targeted metabolomics revealed that Arachidonic acid (AA) metabolism serves as a common pathway between the control and GIOP groups, as well as between the high-dose AU (AUH) and GIOP groups. AU notably upregulates prostaglandin-endoperoxide synthase 2 (PTGS2) and microsomal prostaglandin-E synthase 1 (PTGES) expression and downregulates prostaglandin-H2 D-isomerase (PTGDS) expression. Furthermore, AU treatment increased the expression of runt-related transcription factor 2 (Runx2) and transcription factor Sp7 (Osterix), enhanced serum alkaline phosphatase (ALP) activity, and reduced osteoclast expression. These results indicate that AU is a potential drug for treating GIOP, and its mechanism is related to regulating AA metabolism and promoting osteoblast differentiation. However, the key targets of AU in treating GIOP still need further exploration.


Subject(s)
Arachidonic Acid , Glucocorticoids , Iridoid Glucosides , Metabolomics , Osteoblasts , Osteoporosis , Animals , Mice , Osteoporosis/drug therapy , Osteoporosis/chemically induced , Osteoporosis/metabolism , Female , Arachidonic Acid/metabolism , Glucocorticoids/adverse effects , Metabolomics/methods , Iridoid Glucosides/pharmacology , Osteoblasts/drug effects , Osteoblasts/metabolism , Cell Differentiation/drug effects , Disease Models, Animal , Osteoclasts/drug effects , Osteoclasts/metabolism , Mice, Inbred C57BL , Calcium/metabolism , Eucommiaceae/chemistry , X-Ray Microtomography/methods
18.
Differentiation ; 138: 100789, 2024.
Article in English | MEDLINE | ID: mdl-38896972

ABSTRACT

Osteoclast (OC) differentiation, vital for bone resorption, depends on osteoclast and precursor fusion. Osteoprotegerin (OPG) inhibits osteoclast differentiation. OPG's influence on fusion and mechanisms is unclear. Osteoclasts and precursors were treated with OPG alone or with ATP. OPG significantly reduced OC number, area and motility and ATP mitigated OPG's inhibition. However, OPG hardly affected the motility of precusors. OPG downregulated fusion-related molecules (CD44, CD47, DC-STAMP, ATP6V0D2) in osteoclasts, reducing only CD47 in precursors. OPG reduced Connexin43 phosphorylated forms (P1 and P2) in osteoclasts, affecting only P2 in precursors. OPG disrupted subcellular localization of CD44, CD47, DC-STAMP, ATP6V0D2, and Connexin43 in both cell types. Findings underscore OPG's multifaceted impact, inhibiting multinucleated osteoclast and mononuclear precursor fusion through distinct molecular mechanisms. Notably, ATP mitigates OPG's inhibitory effect, suggesting a potential regulatory role for the ATP signaling pathway. This study enhances understanding of intricate processes in osteoclast differentiation and fusion, offering insights into potential therapeutic targets for abnormal bone metabolism.


Subject(s)
Adenosine Triphosphate , Cell Differentiation , Osteoclasts , Osteoprotegerin , Osteoprotegerin/metabolism , Osteoprotegerin/genetics , Osteoclasts/metabolism , Osteoclasts/cytology , Animals , Adenosine Triphosphate/metabolism , Mice , Connexin 43/metabolism , Connexin 43/genetics , Cell Fusion , CD47 Antigen/metabolism , CD47 Antigen/genetics , Hyaluronan Receptors/metabolism , Hyaluronan Receptors/genetics , Membrane Proteins/metabolism , Membrane Proteins/genetics , Bone Resorption/metabolism , Bone Resorption/genetics , Bone Resorption/pathology , Signal Transduction , Vacuolar Proton-Translocating ATPases/metabolism , Vacuolar Proton-Translocating ATPases/genetics , Nerve Tissue Proteins
19.
Toxicol Lett ; 398: 127-139, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38914176

ABSTRACT

Increasing epidemiological evidence has shown that PM2.5 exposure is significantly associated with the occurrence of osteoporosis. It has been well demonstrated that PM2.5 exposure enhanced the differentiation and function of osteoclasts by indirectly causing chronic inflammation, while the mechanism in osteoblasts remains unclear. In our study, toxic effects were evaluated by direct exposure of 20-80 µg/ml PM2.5 to MC3T3-E1 cells and BMSCs. The results showed that PM2.5 exposure did not affect cell viability via proliferation and apoptosis, but significantly inhibited osteoblast differentiation in a dose-dependent manner. Osteogenic transcription factors Runx2 and Sp7 and other biomarkers Alp and Ocn decreased after PM2.5 exposure. RNA-seq revealed TGF-ß signaling was involved in PM2.5 exposure inhibited osteoblast differentiation, which led to P-Smad1/5 and P-Smad2 reduction in the nucleus by increasing the ubiquitination and degradation of Smad4. At last, the inflammation response increased in MC3T3-E1 cells with PM2.5 exposure. Moreover, the mRNA levels of Mmp9 increased in bone marrow-derived macrophage cells treated with the conditional medium collected from MC3T3-E1 cells exposed to PM2.5. Overall, these results indicated that PM2.5 exposure inhibits osteoblast differentiation and concurrently increases the maturation of osteoclasts. Our study provides in-depth mechanistic insights into the direct impact of PM2.5 exposure on osteoblast, which would indicate the unrecognized role of PM2.5 on osteoporosis.


Subject(s)
Cell Differentiation , Osteoblasts , Particulate Matter , Smad4 Protein , Ubiquitination , Osteoblasts/drug effects , Osteoblasts/metabolism , Animals , Cell Differentiation/drug effects , Smad4 Protein/metabolism , Smad4 Protein/genetics , Mice , Particulate Matter/toxicity , Ubiquitination/drug effects , Signal Transduction/drug effects , Osteogenesis/drug effects , Osteoclasts/drug effects , Osteoclasts/metabolism , Air Pollutants/toxicity , Cell Line , Cell Survival/drug effects , Transforming Growth Factor beta/metabolism , Core Binding Factor Alpha 1 Subunit/metabolism , Core Binding Factor Alpha 1 Subunit/genetics , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase 9/genetics , Proteolysis/drug effects
20.
Cell Mol Biol (Noisy-le-grand) ; 70(6): 217-223, 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38836657

ABSTRACT

Mesenchymal stem cells from bone marrow, such as bone marrow aspirate concentrate (BMAC) and cultured and isolated bone marrow mesenchymal stem cells (BM-MSCs), have been used as therapeutic alternatives to enhance remodeling in the bone. OBJECTIVE: This study aimed to evaluate the effects of BMAC and BM-MSCs on orthodontic tooth movements in rabbits. METHODS: A100- gram nickel-titanium closed-coil springs were used to initiate orthodontic tooth movement of the lower first premolars in 35 male New Zealand rabbits for 21 days. Using a split-mouth design, autologous BMAC or BM-MSCs were submucosally injected into the right sides of the lower jaw, while the left sides served as the control. On days 7, 14, and 21, a three-dimensional digital model scan was used to measure the amount of tooth movement. The microfocus computed tomography (Micro-CT) and histological findings were examined on day 0 as the baseline measurement and on days 7, 14, and 21. RESULTS: Compared to the control group, the quadrant receiving BMAC and BM-MSCs had a considerably greater amount of tooth movement. Histomorphometric analysis revealed that both BMAC and BM-MSCs had significantly higher numbers of osteoclasts and active bone-resorptive lacunae. The resorptive changes were greater in the BMAC and BM-MSCs groups than in the control group. CONCLUSION: The submucosal injection of BMAC and BM-MSCs accelerates orthodontic tooth movement (OTM) by decreasing bone density and supplying more osteoclast progenitor cells.


Subject(s)
Bone Marrow Cells , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Osteoclasts , Tooth Movement Techniques , X-Ray Microtomography , Animals , Rabbits , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Male , Tooth Movement Techniques/methods , Mesenchymal Stem Cell Transplantation/methods , Bone Marrow Cells/cytology , Osteoclasts/cytology , Osteoclasts/metabolism
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