Fhl2 promotes chondrocyte ferroptosis in osteoarthritis by impairing mitophagic flux through microtubule disorganization.

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.