Zinc-MTF1-metallothionein axis plays critical roles in the defense against ferroptosis in human cells

Ferroptosis, an iron-dependent form of regulated cell death characterized by lipid hydroperoxide accumulation, has attracted considerable attention because of its potential clinical applications. Ferroptosis is governed by redox reactions; however, the factors regulating this process, as well as their relationship with the central mediator of cellular redox homeostasis glutathione (GSH), warrant further investigation. Metallothioneins (MTs), cysteine-rich proteins that play central roles in zinc storage and homeostasis, comprise eleven isoforms in humans. This isoform multiplicity complicates functional analysis of human MTs, making it difficult to generate knockout cell lines and elucidate their precise roles in biological processes, including ferroptosis. Here, we established MT-knockout human cell lines and demonstrated that MTs protected against iron overload- or GSH depletion-induced ferroptosis in cooperation with GSH through their abundant thiol groups. Zinc enhanced this protective effect by inducing MT expression via metal-responsive transcription factor 1 (MTF1) activation. Importantly, this protective function was conserved across different human cell types, and MT expression levels may contribute to cell-type-specific susceptibility to ferroptosis. These findings highlight the zinc-MTF1-MT axis as a critical regulatory pathway that complements the GSH-glutathione peroxidase 4 system in controlling ferroptosis and may constitute a promising target for ferroptosis-directed therapeutic strategies.