Background: Proper mitochondrial metabolism is essential for osteoclastogenesis and bone loss. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is an essential enzyme of mitochondrial one-carbon metabolism, catalyzing the interconversion of 5,10-methylene-THF to 10-formyl-THF and generating formate for de novo purine synthesis. Previous reports showed that genetic variations of 5,10-methylenetetrahydrofolate reductase, a critical enzyme for intracellular folate homeostasis, linked to increased susceptibility to RA. Notably, MTHFD2 is highly expressed in cancer cells and immune cells while is ordinarily low or absent in normal adult tissues. Given the narrower range of cells that express this enzyme, MTHFD2 is an ideal target for treating cancer and autoimmune diseases. However, the roles of MTHFD2 in osteoclastogenesis and bone resorption remain unknown.

Objectives: To elucidate the effect and underlying mechanisms of MTHFD2 on osteoclast differentiation and bone loss in rheumatoid arthritis (RA).

Methods: The expression of MTHFD2 was examined in CD14+ monocytes and murine bone marrow derived macrophages (BMMs). RNA-sequencing was performed to evaluate the regulatory mechanisms of MTHFD2 on osteoclastogenesis. Extracellular flux assay, JC-1 staining and transmission electron microscopy were used to detect mitochondrial function and energy metabolism changes during osteoclast formation. Collagen-induced arthritis (CIA) mice were used to evaluate the therapeutic effect of MTHFD2 knockdown on bone loss. Bone volume and osteoclast counts were quantified by μCT and histomorphometry.

Results: Elevated MTHFD2 was observed in RA patients and CIA mice with a positive correlation to bone resorption parameters. During osteoclast formation, MTHFD2 was significantly upregulated in both human CD14+ monocytes and murine BMMs. The application of MTHFD2 inhibitor and MTHFD2 knockdown suppressed osteoclastogenesis, while MTHFD2 overexpression promoted osteoclast differentiation in vitro. RNA-sequencing revealed that MTHFD2 inhibition blocked oxidative phosphorylation (OXPHOS) in osteoclasts, leading to decreased adenosine triphosphate (ATP) production and mitochondrial membrane potential without affecting mitochondrial biogenesis. Mechanistically, inhibition of MTHFD2 downregulated the expression of mitochondrial creatine kinase 1 (CKMT1), which in turn affected phosphocreatine energy shuttle and OXPHOS during osteoclastogenesis. Further, a therapeutic strategy to knockdown MTHFD2 in knee joint in vivo ameliorated bone loss in CIA mice.

Conclusion: Our findings demonstrate that MTHFD2 is upregulated in RA with relation to joint destruction. MTHFD2 promotes osteoclastogenesis and arthritic bone erosion by enhancing mitochondrial energy metabolism through CKMT1. Thus, targeting MTHFD2 may provide a potential new therapeutic strategy for tackling osteoclastogenesis and bone loss in RA.

REFERENCES: [1] Sugiura A, Andrejeva G, Voss K, Heintzman DR, Xu X, Madden MZ, et al. MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function. Immunity. 2022;55(1):65-81 e9.

[2] He L, Lin J, Lu S, Li H, Chen J, Wu X, et al. CKB Promotes Mitochondrial ATP Production by Suppressing Permeability Transition Pore. Adv Sci (Weinh). 2024;11(31):e2403093.

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.