Chronic low-dose iron exposure induces GPX4-mediated resistance to ferroptosis in a human chondrocyte cell line

Osteoarthritis (OA) is a degenerative joint disease closely linked to iron dysregulation. While the role of iron in OA is recognized, the precise mechanism of cell death remains unclear. This study aimed to elucidate how chronic low-dose iron exposure influences chondrocyte survival, iron metabolism, and ferroptosis susceptibility. The 32-day exposure effects of low-dose (20 µM) ferric ammonium citrate (FAC) treatment on the immortalized human chondrocyte cell line (C-20/A4) in terms of survival and susceptibility to ferroptosis were investigated. Cell viability, morphology, and expression of key iron regulatory genes (IRGs) and ferroptosis-related stress genes were assessed. At day-32 of FAC treatment, chondrocyte proliferation and glutathione peroxidase 4 (GPX4) expression increased relative to controls (p < 0.0001). Inhibition of GPX4 with RSL3 in FAC-treated cells resulted in significantly increased cell death (p < 0.001), indicating a possible protective role of GPX4. Iron chelation with deferoxamine conferred protection against ferroptosis. Dynamic changes in IRGs and cell cycle regulators, consistent with an adaptive strategy to resist iron overload and support survival, were observed. In-silico analysis of OA cartilage transcriptomic data identified 170 differentially expressed genes. Functional enrichment showed upregulation of ubiquitin-dependent catabolic processes and downregulation of mitotic pathways, supporting altered stress responses and cell cycle disruptions in iron-exposed chondrocytes. Chronic low-dose iron exposure induced an adaptive upregulation of GPX4, enhancing chondrocyte resistance to cell death by ferroptosis by maintaining lipid peroxide reduction. Therapeutic modulation of GPX4 and iron homeostasis may offer novel strategies for OA management where cartilage iron imbalance is implicated.