Gadd45a knockout alleviates cisplatin-induced hearing loss by inhibiting CXCL family protein expression.

Abstract

Background: Cisplatin is a widely used chemotherapeutic agent, but its clinical application is limited by serious ototoxic side effects, including hearing loss, tinnitus, and vertigo. Inflammation is now recognized as a significant contributor to cisplatin-induced hearing loss, but the upstream regulators remain largely undefined. This study aimed to identify novel regulators of cisplatin-induced ototoxicity through CRISPR-Cas9 screening and to determine the role of growth arrest and DNA damage-inducible alpha (GADD45A) in cochlear inflammation and hearing loss.

Methods: To identify upstream regulators of cisplatin-induced ototoxicity, a CRISPR-Cas9-based loss-of-function screen was first performed in OC1 cells. Following the identification of Gadd45a as a candidate gene, its effects on cisplatin-induced cytotoxicity were evaluated using cell viability assays, flow cytometry, and TUNEL staining. Integrated transcriptomic and proteomic analyses were subsequently conducted to elucidate the downstream molecular mechanisms. Finally, the protective role of Gadd45a inhibition was validated in vivo through siRNA delivery into the posterior semicircular canal and in Gadd45a conditional knockout (cKO) mice.

Results: Gadd45a knockout significantly inhibited cisplatin-induced cell death in the OC1 cell line while simultaneously enhancing autophagy signaling. Transcriptomic profiling revealed a marked downregulation of the expression of C-X-C motif chemokine ligand (CXCL) family cytokines following Gadd45a loss, which was further supported by proteomic analysis of culture supernatants. Mechanistically, Gadd45a knockout specifically inhibited nuclear factor κB subunit 1 (NF-κB1) but not RELA proto-oncogene, NF-κB subunit (RELA). Immunofluorescence staining demonstrated that Gadd45a knockout inhibited the nuclear translocation of the NF-κB1 protein, and subsequent studies revealed that the NF-κB1 protein was degraded through the lysosomal pathway. In vivo, both si-Gadd45a injection into the semicircular canal and the knockout of Gadd45a effectively attenuated cisplatin-induced ototoxicity.

Conclusions: This study identifies GADD45A as a key regulator of cisplatin ototoxicity and links the balance between apoptosis and autophagy with NF-κB1-CXCL-mediated inflammation. Targeted inhibition of GADD45A signaling may represent a promising therapeutic strategy to prevent hearing loss in patients receiving cisplatin chemotherapy.