Cystathionine γ‐lyase‐dependent S‐sulfhydration of Smad3: A novel target to alleviate fibrosis in systemic sclerosis

Objective The cystathionine γ‐lyase (CSE)/H 2 S axis has emerged as a key regulator in tissue fibrogenesis. This study aimed to explore the role of the CSE/H 2 S axis in systemic sclerosis (SSc) and to investigate its underlying mechanisms to identify promising therapeutic targets. Methods CSE/H₂S levels were assessed in serum samples from 25 patients with SSc and 28 healthy controls. Human dermal fibroblasts from SSc patients and healthy controls were used for functional studies, including PAG (CSE inhibitor) treatment, Gyy4137 (H 2 S agent), CSE silencing, and CSE overexpression, combined with LC–MS/MS–based S‐sulfhydration proteomics. Molecular dynamics simulations were performed to study the effects of S‐sulfhydration on protein structure, and a Smad3 C121S (Cys121 mutated to Ser) mutant was generated to verify the function targets of S‐Sulfhydration. In vivo, bleomycin‐induced mouse models of skin and lung fibrosis were constructed to evaluate the effects of CSE overexpression. Results In human samples, CSE/H₂S levels were reduced in SSc. CSE inhibition promoted extracellular matrix deposition. S‐sulfhydration proteomics showed that S‐sulfhydration levels were globally reduced in SSc compared to controls. CSE overexpression increased S‐sulfhydration on Smad3, suppressed TGF‐β1/Smad3 signaling, mitigating skin fibrosis. Notably, cysteine 121 on Smad3, identified as a pivotal target for S‐sulfhydration by proteomics, was shown to fine‐tune its MH1 domain, with its mutation impairing the antifibrotic effects. In mice, CSE overexpression attenuated bleomycin‐induced skin and lung fibrosis. Conclusion Smad3 S‐sulfhydration mediates the antifibrotic effect of CSE in SSc, highlighting it as a critical mechanism and promising therapeutic target.