Poster Presentation 1st Asia Pacific Herbert Fleisch Workshop 2025

Regulation of Bone and Cartilage Metabolism by Supersulfides and Their Therapeutic Potential (#108)

Miki Maemura 1 , Masanobu Morita 2 , Seiryo Ogata 2 , Yoichi Miyamoto 3 , Tomoaki Ida 4 , Kazuhiro Shibusaka 5 , Soichiro Negishi 1 , Masahiro Hosonuma 6 , Taku Saito 7 , Jun Yoshitake 4 , Tsuyoshi Takata 4 , Tetsuro Matsunaga 8 , Eikan Mishima 9 , Uladzimir Barayeu 10 , Takaaki Akaike 4 , Fumiko Yano 11 12
  1. Department of Oral and Maxillofacial Surgery, Graduate School of Dentistry, Showa Medical University, Tokyo
  2. Department of Environmental Medicine and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai
  3. Faculty of Arts and Sciences at Fujiyoshida, Showa Medical University, Fujiyoshida
  4. Department of Redox Molecular Medicine, Tohoku University Graduate School of Medicine, Sendai
  5. Department of Orthodontics, Graduate School of Dentistry, Showa Medical University, Tokyo
  6. Department of Pharmacology, Showa Medical University, Tokyo
  7. Sensory & Motor System Medicine，Graduate School of Medicine, The University of Tokyo, Tokyo
  8. Centre for Integrated Control, Epidemiology and Molecular Pathophysiology of Infectious Diseases, Akita University, Akita
  9. Institute of Metabolism and Cell Death, Molecular Targets and Therapeutics Center, Helmholtz Munich
  10. Max-Planck-Institute for Polymer Research, Mainz
  11. Showa Medical University Research Administration Center, Showa Medical University, Tokyo
  12. Department of Biochemistry, Graduate School of Dentistry, Showa Medical University, Tokyo

Background:
Polysulfides, a class of molecules containing multiple sulfur atoms, have recently been identified as biologically active species involved in alternative sulfur respiration and other physiological functions. This study aimed to investigate the role of cysteinyl-tRNA synthetase 2 (CARS2), a key enzyme for polysulfide production, in bone and cartilage metabolism.

Methods:
We employed a tibial fracture model using Cars2 mutant mice (Cars2AINK/+) and wild-type littermates (Cars2+/+). Histological analysis, bulk RNA-seq, and qRT-PCR were performed on the fracture callus at 2 weeks post-operation. In an osteoarthritis (OA) model, histological evaluation was conducted 16 weeks after surgery, and intra-articular administration of the polysulfide molecule GSSSG was assessed. In vitro, primary mouse synovial fibroblasts were treated with IL-1β and polysulfides, followed by qRT-PCR. Sulfur metabolomics using LC-ESI-MS/MS was performed on synovial and cartilage tissues from both genotypes. Uptake of GSSSG was evaluated in ATDC5 cells using stable isotope-labeled GSSSG.

Results:
Cars2AINK/+ mice showed delayed callus formation in the fracture model. Transcriptomic analyses revealed upregulation of inflammatory SASP-related genes and downregulation of endochondral ossification markers. In the OA model, disease progression was significantly accelerated in Cars2AINK/+ mice. Conversely, administration of 30 µM GSSSG significantly suppressed OA progression in wild-type mice. GSSSG also reduced IL-1β-induced inflammatory gene expression in synovial fibroblasts. Sulfur metabolomics indicated decreased levels of reactive sulfur species and polysulfide metabolites in Cars2AINK/+ synovium and cartilage. In ATDC5 cells, only GSSSG treatment elevated intracellular reactive sulfur levels, with isotope tracing confirming cellular uptake of exogenous sulfur from labeled GSSSG.

Conclusion:
CARS2-derived polysulfides, particularly GSSSG, may suppress OA-related inflammation and lipid peroxidation, potentially by inhibiting ferroptosis. This study highlights the anti-inflammatory and antioxidant roles of polysulfides in the musculoskeletal system and suggests their therapeutic relevance in fracture healing and OA treatment.