Ferroptosis contributes to osteoarthritis (OA), but the upstream mechanisms connecting cytoskeletal stress, mitochondrial quality control, and antioxidant defense remain unclear. We investigated whether four-and-a-half LIM domain protein 2 (Fhl2) couples microtubule remodeling to chondrocyte ferroptosis. Public bulk and single-cell transcriptomes were integrated with RNA sequencing (RNA-seq) from RSL3-treated chondrocytes to nominate ferroptosis regulators in OA. Fhl2 was validated in human cartilage, destabilization of the medial meniscus (DMM) mouse cartilage, and interleukin-1 beta (IL-1β)- or RSL3-treated primary murine chondrocytes. Lentiviral Fhl2 knockdown, microtubule imaging, mitochondria-lysosome co-localization, mitophagy marker analysis, paclitaxel/nocodazole perturbation, intra-articular sh-Fhl2 delivery, and cartilage-specific nuclear factor erythroid 2-related factor 2 (Nrf2) conditional knockout were used to define mechanism and in vivo relevance. Fhl2 emerged as a shared hub across OA bulk signatures, chondrocyte states prone to ferroptosis, and the RSL3-induced ferroptosis program, and was elevated in human OA cartilage, DMM cartilage, and stressed chondrocytes. Fhl2 upregulation accompanied microtubule disorganization, impaired mitophagic flux, translocase of outer mitochondrial membrane 20 (TOMM20)/p62/microtubule-associated protein 1 light chain 3 beta (LC3B) accumulation, mitochondrial retention, reactive oxygen species (ROS) production, and lipid peroxidation. Fhl2 knockdown preserved microtubules, improved mitochondria-lysosome coupling, reduced ferroptotic injury, attenuated cartilage degeneration after DMM, improved gait parameters, and restored Nrf2/glutathione peroxidase 4 (GPX4) signaling. Nrf2 deficiency was associated with matrix catabolism and a ferroptosis-prone cartilage phenotype after DMM. Fhl2 promotes chondrocyte ferroptosis in OA by linking impaired microtubule-dependent mitophagic flux to mitochondrial retention and antioxidant failure. Targeting the link between Fhl2, cytoskeletal integrity, and mitochondrial quality control may help limit cartilage degeneration driven by ferroptosis.
