Dataset Information

Protein Succinylome analysis identifies citrate synthase as a central regulator of osteoclast metabolic activity

ABSTRACT: The cytokines tumor necrosis factor ligand superfamily member 11 (TNFSF11; also known as RANKL) and macrophage colony-stimulating factor 1 receptor (M-CSF) differentiate macrophages into bone-resorbing osteoclasts via a process characterised by changes in metabolic activity that support energy-consuming processes such as cell fusion or bone resorption. Treatment with RANKL triggers a phenotype of accelerated metabolism with enhanced glycolysis and an initial disruption of the tricarboxylic acid cycle (TCA) cycle through increased expression of the enzyme aconitate decarboxylase (ACOD1). which results in an upregulation of intracellular succinate levels. Succinate then causes post-translational succinylation of lysine residues. Interestingly, ACOD1 as an inducer of protein succinylation and the desuccinylase NAD-dependent protein deacylase sirtuin-5, mitochondrial (SIRT5) are regulated differentially and the initially high expression of ACOD1 decreases towards the end of differentiation, whereas SIRT5 levels increase. To mimic the effect of protein succinylation, diethyl succinate or a SIRT5 inhibitor were added to differentiating osteoclasts, which reduced the formation of large osteoclasts, showing its relevance for successful osteoclastogenesis. To identify proteins succinylated after RANKL treatment, we used an immunoaffinity-based liquid chromatography–tandem mass spectrometry (LC-MS/MS) approach. Most lysine succinylated proteins were metabolic enzymes localised in the mitochondria. Citrate synthase, the enzyme catalysing the first reaction of the TCA cycle, showed a notable difference in succinylation levels before and after RANKL stimulation, with succinylation detected exclusively in stimulated cells. Immunoprecipitation assays confirmed citrate synthase succinylation. Using whole cell extracts, we observed that RANKL treatment decreased CS activity in a concentration-dependent manner. This suggests that CS could be a critical factor in the context of energy production during osteoclastogenesis and that protein succinylation helps to modulate the differentiation program of osteoclasts.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Macrophage

SUBMITTER: Marcin Luzarowski

LAB HEAD: Marcin Luzarowski

PROVIDER: PXD060675 | Pride | 2025-07-21

REPOSITORIES: Pride

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Protein succinylome analysis identifies citrate synthase as a central regulator of osteoclast metabolic activity.

Yu Dayoung D Gao Yue Y Luzarowski Marcin M Seebach Elisabeth E Heitkamp Thomas T Börsch Michael M Ruppert Thomas T Kubatzky Katharina F KF

The FEBS journal 20250402 14

Tumour necrosis factor ligand superfamily member 11 (TNFSF11; RANKL) and macrophage colony-stimulating factor 1 receptor (M-CSF) differentiate macrophages into osteoclasts. This process is characterised by changes in metabolic activity that support energy-consuming processes. Treatment with RANKL triggers a phenotype of accelerated metabolism with enhanced glycolysis and an initial disruption of the tricarboxylic acid cycle (TCA) through increased expression of the enzyme aconitate decarboxylase ...[more]

PMID: 40172090

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Project description:Purpose: Among the diverse cytokines involved in osteoclast differentiation, IL-3 has been shown to inhibit RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. In the present study, we demonstrate that IL-3 activation of STAT5 inhibits RANKL-induced osteoclastogenesis through the induction of Id genes. Methods: To investigate the effect of STAT5 on osteoclast differentiation and IL-3-mediated inhibition of osteoclast differentiation, bone marrow derived macrophages isolated from STAT5 wild-type (Stat5fl/fl) or STAT5 cKO (STAT5;MX1-Cre) were differentiated to osteoclast in the presence of M-CSF and RANKL with or without IL-3; and bone marrow derived macrophges from STAT5 wild-type and STAT5 cKO were overexpressed with PMX-FIG (control) or STAT5A1*6 (constitutively active form of STAT5A) and differentiated to osteoclast. To analyze bone phenotype, femurs and tibiae of 16 week-old STAT5 wild-type and STAT5 cKO were subjected to micro CT analysis and histomorphometry, respectively. Results: Overexpression of STAT5 inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate either expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting that STAT5 plays an important role in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated expression of the Id1 and Id2 genes, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Moreover, micro-computed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in STAT5 conditional knockout mice than in wild-type mice in a RANKL injection model. Conclusion: Taken together, our results suggest that STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis through Id gene expression. Examination of 4 different combination of osteoclast differentiation condition of bone marrow derived macrophages.

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	Q25070-22-120-noMBR_1.msf	Msf
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