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TNF-? has both stimulatory and inhibitory effects on mouse monocyte-derived osteoclastogenesis.

ABSTRACT: Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF-?, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31^hi Ly-6C^- ), myeloid blasts (CD31⁺ Ly-6C⁺ ), and monocytes (CD31^- Ly-6C^hi ) were sorted from mouse bone marrow using flow cytometry and cultured with M-CSF and RANKL, with or without TNF-?. Surprisingly, TNF-? prevented the differentiation of TRAcP⁺ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL-1? or IL-6. When monocytes were pre-cultured with M-CSF and RANKL followed by exposure to TNF-?, a stimulatory effect was found. TNF-? also stimulated monocytes' osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF-? was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down-regulated while TNF-?R1 and TNF-?R2 were up-regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF-?, indicating an altered ratio of bound-RANK/unbound-RANK. Our findings suggest a diverse role of TNF-? on monocytes' osteoclastogenesis: it affects the RANK-signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre-cultured with M-CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte-derived osteoclast formation away from the bone.

SUBMITTER: Cao Y

PROVIDER: S-EPMC5601245 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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TNF-α has both stimulatory and inhibitory effects on mouse monocyte-derived osteoclastogenesis.

Cao Yixuan Y Jansen Ineke D C IDC Sprangers Sara S de Vries Teun J TJ Everts Vincent V

Journal of cellular physiology 20170717 12

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF-α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31hi Ly-6C- ), myeloid blasts (CD31+ Ly-6C+ ), and monocytes (CD31- Ly-6Chi ) were sorted from mouse bone marrow using flow cytometry and cul ...[more]

PMID: 28543070

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Project description:Increased osteoclastogenesis is responsible for osteolysis, which is a severe consequence of inflammatory diseases associated with bone destruction, such as rheumatoid arthritis and periodontitis. The mechanisms that limit osteoclastogenesis under inflammatory conditions are largely unknown. We previously identified transcription factor RBP-J as a key negative regulator that restrains TNF-α-induced osteoclastogenesis and inflammatory bone resorption. In this study, we tested whether RBP-J suppresses inflammatory osteoclastogenesis by regulating the expression of microRNAs (miRNAs) important for this process. Using high-throughput sequencing of miRNAs, we obtained the first, to our knowledge, genome-wide profile of miRNA expression induced by TNF-α in mouse bone marrow-derived macrophages/osteoclast precursors during inflammatory osteoclastogenesis. Furthermore, we identified miR-182 as a novel miRNA that promotes inflammatory osteoclastogenesis driven by TNF-α and whose expression is suppressed by RBP-J. Downregulation of miR-182 dramatically suppressed the enhanced osteoclastogenesis program induced by TNF-α in RBP-J-deficient cells. Complementary loss- and gain-of-function approaches showed that miR-182 is a positive regulator of osteoclastogenic transcription factors NFATc1 and B lymphocyte-induced maturation protein-1. Moreover, we identified that direct miR-182 targets, Foxo3 and Maml1, play important inhibitory roles in TNF-α-mediated osteoclastogenesis. Thus, RBP-J-regulated miR-182 promotes TNF-α-induced osteoclastogenesis via inhibition of Foxo3 and Maml1. Suppression of miR-182 by RBP-J serves as an important mechanism that restrains TNF-α-induced osteoclastogenesis. Our results provide a novel miRNA-mediated mechanism by which RBP-J inhibits osteoclastogenesis and suggest that targeting of the newly described RBP-J-miR-182-Foxo3/Maml1 axis may represent an effective therapeutic approach to suppress inflammatory osteoclastogenesis and bone resorption.

Project description:BackgroundEnhancer of zeste homolog 2 (EZH2) is a novel oncogene that can specifically trimethylate the histone H3 lysine 27 (H3K27me3) to transcriptionally inhibit the expression of downstream tumor-suppressing genes. As a small molecular inhibitor of EZH2, 3-Deazaneplanocin (DZNep) has been widely studied due to the role of tumor suppression. With the roles of epigenetic regulation of bone cells emerged in past decades, the property and molecular mechanism of DZNep on enhancing osteogenesis had been reported and attracted a great deal of attention recently. This study aims to elucidate the role of DZNep on EZH2-H3K27me3 axis and downstream factors during both osteoclasts and osteoblasts formation and the therapeutic possibility of DZNep on bone defect healing.MethodsBone marrow-derived macrophages (BMMs) cells were cultured, and their responsiveness to DZNep was evaluated by cell counting kit-8, TRAP staining assay, bone resorption assay, podosome actin belt. Bone marrow-derived mesenchymal stem cells (BMSC) were cultured and their responsiveness to DZNep was evaluated by cell counting kit-8, ALP and AR staining assay. The expression of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), Wnt signaling pathway was determined by qPCR and western blotting. Mouse bone defect models were created, rescued by DZNep injection, and the effectiveness was evaluated by X-ray and micro-CT and histological staining.ResultsConsistent with the previous study that DZNep enhances osteogenesis via Wnt family member 1(Wnt1), Wnt6, and Wnt10a, our results showed that DZNep also promotes osteoblasts differentiation and mineralization through the EZH2-H3K27me3-Wnt4 axis. Furthermore, we identified that DZNep promoted the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation via facilitating the phosphorylation of IKKα/β, IκB, and subsequently NF-κB nuclear translocation, which credit to the EZH2-H3K27me3-Foxc1 axis. More importantly, the enhanced osteogenesis and osteoclastogenesis result in accelerated mice bone defect healing in vivo.ConclusionDZNep targeting EZH2-H3K27me3 axis facilitated the healing of mice bone defect via simultaneously enhancing osteoclastic bone resorption and promoting osteoblastic bone formation.

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TNF-? has both stimulatory and inhibitory effects on mouse monocyte-derived osteoclastogenesis.

Publications

TNF-α has both stimulatory and inhibitory effects on mouse monocyte-derived osteoclastogenesis.

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