ETS1-driven transcriptional activation of ACSL4 mediates hypoxia-induced ferroptosis in anterior cruciate ligament fibroblasts

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Background Anterior cruciate ligament (ACL) injury is a prevalent sports-related trauma that severely compromises knee joint function. The hypoxic microenvironment is a critical pathological driver of fibroblast dysfunction following ACL injury; however, whether ferroptosis participates in this process and the underlying regulatory mechanisms remain elusive. Methods Primary ACL fibroblasts were isolated and cultured under hypoxic conditions. Cell viability, proliferation, apoptosis, and invasive capacity were evaluated by cell counting kit-8, 5-Ethynyl-2′-deoxyuridine, flow cytometry, and Transwell assays, respectively. Ferroptosis involvement was validated using ferrostatin-1 and erastin. Expression levels of the ferroptosis-related protein Acyl-CoA synthetase long-chain family member 4 (ACSL4) were determined by Western blot. Bioinformatic prediction and experimental verification, including dual-luciferase reporter and chromatin immunoprecipitation assays, were employed to investigate ETS proto-oncogene 1 (ETS1)-mediated transcriptional regulation of ACSL4. Results Hypoxia significantly suppressed ACL fibroblast viability, proliferation, and invasion while inducing apoptosis (all P < 0.01). Hypoxic cells exhibited ferroptotic features, including elevated Fe 2+ and malondialdehyde, depleted glutathione, and increased lipid reactive oxygen species, accompanied by ACSL4 upregulation ( P < 0.001). ACSL4 silencing attenuated hypoxia-induced ferroptosis and cellular dysfunction. Mechanistically, hypoxia upregulated ETS1 expression ( P < 0.01), which directly bound the ACSL4 promoter to activate its transcription. Forced ACSL4 expression reversed the protective effects of ETS1 knockdown on hypoxia-induced injury and ferroptosis. Conclusion Hypoxia induces ferroptosis in ACL fibroblasts. The transcription factor ETS1 promotes this process by directly activating ACSL4 transcription. The ETS1-ACSL4 axis represents a potential therapeutic target for ACL injury.

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