Discussion

The present study identifies GADD45A as a critical mediator of cisplatin-induced ototoxicity. Through CRISPR-Cas9-mediated gene knockout, siRNA-mediated knockdown, cKO mouse models, we demonstrated that Gadd45a deficiency attenuated cisplatin-induced cochlear hair cell loss and hearing impairment. Mechanistically, Gadd45a deficiency reduced NF-κB1 protein levels, suppressed the expression of CXCL family chemokines, enhanced autophagic activity, attenuated apoptotic signaling, and ultimately preserved cochlear structural integrity and hearing function (Fig. 7). These findings suggest that GADD45A contributes to cisplatin-induced ototoxicity through coordinated regulation of stress responses, inflammation, and cell survival pathways.

Fig. 7: Schematic representation of the mechanism of siGadd45a-mediated protection of hair cells from cisplatin damage. Cisplatin treatment induces Gadd45a expression, which promotes NF-κB1 activation and CXCL family-mediated inflammatory responses, leading to hair cell apoptosis. Silencing of GADD45A suppresses NF-κB1/CXCL signaling and attenuates inflammation and apoptosis, thereby protecting hair cells from cisplatin-induced injury. SiGadd45a. Small interfering RNA targeting Gadd45a; OHC. Outer hair cell; GADD45A. Growth arrest and DNA damage-inducible alpha; NF-κB1. Nuclear factor kappa B subunit 1; CXCL. C-X-C motif chemokine ligand; JNK. c-Jun N-terminal kinase. PSCC. Posterior semicircular canal.

The fluctuation of platinum tolerance-related gene expression during 24 h of cisplatin treatment indicates that modulating the expression of these genes may mitigate cisplatin-induced ototoxicity [14], [45], [48]. Although manipulation of Top2a [46], [48] and Birc5 [45] did not produce significant protection in our experimental models, genetic ablation of Gadd45a markedly attenuated cisplatin-induced cochlear damage, identifying Gadd45a as a key regulator of cisplatin ototoxicity. Previous studies have primarily linked GADD45A to DNA damage responses and stress-induced apoptosis through activation of the MKK4-JNK signaling pathway [29], [51]. Consistent with these reports, Gadd45a expression was markedly induced following cisplatin exposure in our study. Notably, Gadd45a deficiency significantly reduced TUNEL+ cells, suggesting that GADD45A contributes to cisplatin-induced apoptotic signaling and subsequent cell death. These findings further extend the current understanding of GADD45A by the protective effect of Gadd45a deficiency in cochlear hair cells during cisplatin-induced ototoxicity.

Time-course cisplatin treatment experiments were subsequently performed to investigate the mechanisms underlying cisplatin tolerance. The temporal dynamics of the expression of DNA damage markers, particularly γ-H2A.X, revealed critical insights into the protective mechanism of Gadd45a knockout. These findings align with our observations that autophagic flux is enhanced, which may facilitate the clearance of cisplatin-DNA adducts through alternative repair mechanisms. The conserved nature of this response across tumor and cochlear cell types highlights the evolutionary importance of GADD45A-mediated stress adaptation. Previous studies have shown that activation of autophagy protects cochlear hair cells from cisplatin-induced damage by reducing oxidative stress and apoptosis [52], [53]. Our findings are consistent with these observations and suggest that enhanced autophagic flux may contribute to the protective phenotype observed in Gadd45a-deficient cells.

To further elucidate the mechanisms underlying GADD45A-mediated cisplatin tolerance, we conducted transcriptomic sequencing. Transcriptomic profiling revealed a striking downregulation of the expression of chemokine signaling components (CXCL1/3/10/12) in Gadd45a-deficient cells, implicating inflammatory regulation as a novel facet of Gadd45a biology. This finding was further supported by reduced NF-κB1 expression and decreased CXCL1/3/10 protein levels in both cultured cells and cochlear tissues, indicating that Gadd45a deficiency suppresses inflammatory signaling. Inflammatory signaling has been increasingly recognized as a key contributor to cisplatin-induced ototoxicity. Prior research has demonstrated that inhibiting CXCL1 effectively reduces cisplatin ototoxicity [25]. Moreover, the increase in inflammatory mediators, such as TNF-α, was attenuated by Stat1 siRNA, and cells were protected from cisplatin-mediated apoptosis due to this inhibition [54]. The downregulation of NF-κB expression by flunarizine through the activation of the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway has been shown to reduce cisplatin-induced ototoxicity, thereby inhibiting the production of proinflammatory cytokines both in vitro and in vivo [55].

Previous research has shown that the homeostasis of the NF-κB family is intricately linked to the expression levels of inflammatory factors [56]. We identified NF-κB1 (p105/p50) and RELA (p65) as potential targets using transcription factor prediction and scoring software. We next explored the relationship between GADD45A and NF-κB. GADD45A may be regulated by NF-κB1, which mediates the activation of the GADD45A-MKK4-JNK cascade and apoptosis [29]. However, the role of this transcription factor in modulating GADD45A expression remains controversial. In contrast, our findings suggest that GADD45A can also regulate NF-κB1 stability and activity, indicating a potentially bidirectional interaction between these pathways. NF-κB1 expression decreased while RELA increased when Gadd45a was knocked out, demonstrating that this chemokine suppression occurs through NF-κB1-dependent mechanisms rather than through canonical RELA pathways. The differential regulation of NF-κB subunits suggests a previously unrecognized specificity in GADD45A-mediated inflammatory control, potentially explaining the broad-spectrum anti-inflammatory effects observed in the knockout models.

Since GADD45A is involved in the regulation of inflammatory factors, which are typically produced by macrophages, we investigated the cellular sources of cochlear inflammation. In the normal physiological state of the cochlea, resident macrophages are present in a dormant state within the cochlear tissue. Upon exposure to cisplatin, these resident macrophages become activated, resulting in the release of inflammatory mediators. This activation, in turn, stimulates peripheral immune cells to infiltrate damaged areas of the cochlea, thereby amplifying the inflammatory response [50]. OC1 is an immortalized cell line, originating from the cochlear sensory epithelium. Because CXCL1, CXCL3, and CXCL10 are secreted proteins, BFA was used to inhibit protein secretion and promote their intracellular accumulation before cell lysis. We found that the CXCL1, CXCL3, and CXCL10 proteins were significantly expressed in OC1 cells, with no detectable signal in the Gadd45a knockout group. Immunofluorescence localization studies revealed impaired nuclear translocation of NF-κB1 in knockout cells, indicating that Gadd45a influences both the expression and activation dynamics of this transcription factor. This dual regulatory capacity positions GADD45A as a master coordinator of inflammatory responses to ototoxic stress [56].

Several limitations should be acknowledged. First, although our data support a role for GADD45A in regulating NF-κB1-dependent inflammatory signaling, the precise molecular mechanism by which GADD45A controls NF-κB1 lysosomal degradation requires further investigation. Second, although the molecular mechanisms were extensively investigated in OC1 cells, cell type-specific validation within the cochlea remains necessary. Third, although conditional Gadd45a knockout and siRNA-mediated knockdown both conferred protection against cisplatin ototoxicity, the long-term effects of Gadd45a inhibition on cochlear homeostasis and auditory function remain unknown. Future studies addressing these questions will be important for evaluating the therapeutic potential of targeting GADD45A in hearing loss.