Protein <i>S</i> ‐glutathionylation stimulate adipogenesis by stabilizing C/EBPβ in 3T3L1 cells
Yosuke Watanabe, Kazuhiro Watanabe, Daisuke Fujioka, Kazuto Nakamura, Takamitsu Nakamura, Manabu Uematsu, Markus Bachschmid, Reiko Matsui, Kiyotaka Kugiyama
Abstract
Reactive oxygen species (ROS) increase during adipogenesis and in obesity. Oxidants react with cysteine residues of proteins to form glutathione (GSH) adducts, S-glutathionylation, that are selectively removed by glutaredoxin-1 (Glrx). We have previously reported that Glrx knockout mice had increased protein S-glutathionylation and developed obesity by an unknown mechanism. In this study, we demonstrated that 3T3L1 adipocytes differentiation increased ROS and protein S-glutathionylation. Glrx ablation elevated protein S-glutathionylation and lipid content in 3T3L1 cells. Glrx replenishment decreased the lipid content of Glrx KO 3T3L1 cells. Glrx KO also increased protein expression and protein S-glutathionylation of the adipogenic transcription factor CCAAT enhancer-binding protein (C/EBP) β. Protein S-glutathionylation decreased the interaction of C/EBPβ and protein inhibitor of activated STAT (PIAS) 1, a small ubiquitin-related modifier E3 ligase that facilitates C/EBPβ degradation. Experiments with truncated mutant C/EBPβ demonstrated that PIAS1 interacted with the liver-enriched inhibitory protein (LIP) region of C/EBPβ. Furthermore, mass spectrometry analysis identified protein S-glutathionylation of Cys201 and Cys296 in the LIP region of C/EBPβ. The C201S, C296S double-mutant C/EBPβ prevented protein S-glutathionylation and preserved the interaction with PIAS1. In summary, Glrx ablation stimulated 3T3L1 cell differentiation and adipogenesis via increased protein S-glutathionylation of C/EBPβ, stabilizing and increasing C/EBPβ protein levels.