Loss of STAT1 protects hair cells from ototoxicity through modulation of STAT3, c-Jun,Akt, and autophagy factors

Hair cell damage is a side effect of cisplatin and aminoglycoside use. The inhibition or attenuation of this process is a target of many investigations. There is growing evidence that STAT1 deficiency decreases cisplatin-mediated ototoxicity; however, the role of STAT function and the molecules that act in gentamicin-mediated toxicity have not been fully elucidated. We used mice lacking STAT1 to investigate the effect of STAT1 ablation in cultured organs treated with cisplatin and gentamicin. Here we show that ablation of STAT1 decreased cisplatin toxicity and attenuated gentamicin-mediated hair cell damage. More TUNEL-positive hair cells were observed in explants of wild-type mice than that of STAT1^−/− mice. Although cisplatin increased serine phosphorylation of STAT1 in wild-type mice and diminished STAT3 expression in wild-type and STAT1^−/− mice, gentamicin increased tyrosine phosphorylation of STAT3 in STAT1^−/− mice. The early inflammatory response was manifested in the upregulation of TNF-α and IL-6 in cisplatin-treated explants of wild-type and STAT1^−/− mice. Expression of the anti-inflammatory cytokine IL-10 was altered in cisplatin-treated explants, upregulated in wild-type explants, and downregulated in STAT1^−/− explants. Cisplatin and gentamicin triggered the activation of c-Jun. Activation of Akt was observed in gentamicin-treated explants from STAT1^−/− mice. Increased levels of the autophagy proteins Beclin-1 and LC3-II were observed in STAT1^−/− explants. These data suggest that STAT1 is a central player in mediating ototoxicity. Gentamicin and cisplatin activate different downstream factors to trigger ototoxicity. Although cisplatin and gentamicin triggered inflammation and activated apoptotic factors, the absence of STAT1 allowed the cells to overcome the effects of these drugs.The process of auditory sensorineural damage implicates a variety of intracellular events caused by aging, noise exposure, aminoglycoside antibiotics, or the chemotherapeutic agent cisplatin. The mechanisms underlying the ototoxic effects of cisplatin and gentamicin are not yet completely understood. Their ototoxicity likely involves morphological changes and the modulation of pro- and anti-apoptotic cell responses.¹Activation of oxidative stress and the inflammatory response are common effects of cisplatin- and gentamicin-induced ototoxicity.² Cisplatin increased the early release of pro-inflammatory cytokines in HEI-OC1 cells and in the cochlea of cisplatin-injected rats.³ Similarly, gentamicin induced the production of pro-inflammatory cytokines in the organ of Corti explants in vitro.⁴ The JAK/STAT pathway is one of the best-characterized cellular signaling pathways in the immune system. STAT1, a regulator of cell death, has been reported to be involved in cisplatin-mediated hair cell damage.^{5, 6} Knockdown of the STAT1 gene by means of siRNA, administrated by transtympanic injection in rats and transfection of UB/OC1 cells, reduced cisplatin-induced hair cell death in vivo and in vitro. Moreover, STAT1 siRNA preserved hearing in cisplatin-treated rats.⁵ Furthermore, STAT1 phosphorylation has been observed in utricles exposed to cisplatin in vitro.⁶The inactivation of STAT1 in other tissues has also demonstrated a protective effect, for example, by enhancing autophagy in STAT1-deficient hearts⁷ or accelerated skeletal muscle regeneration.⁸ Recent findings demonstrated that inhibition of the JAK2/STAT3 signaling pathway protects against noise-induced damage to cochlear tissue⁹ and STAT3/SOCS3 signaling regulate hair cell regeneration.¹⁰ Generally, STAT1 and STAT3 are reciprocally regulated, and disruption of their balance directs cells from survival to apoptotic cell death or from inflammatory to anti-inflammatory responses.¹¹ However, there is no information about the role of STAT1 in gentamicin-induced hair cell damage.In the present study, we investigated the impact of the genetic ablation of STAT1 on hair cell damage induced by cisplatin and gentamicin. We also examined a subset of cell signaling mediators involved in apoptosis and survival. Our data indicate that STAT1 has an important role in cisplatin- and gentamicin-mediated hair cell death. We observed differences in the expression of STAT1 and STAT3 in the organ of Corti (OC) from wild-type (WT) and STAT1^−/− mice exposed to cisplatin or gentamicin. An early inflammatory response was observed in the cisplatin-treated explants. Finally, we demonstrated regulatory changes of Akt, c-Jun, and autophagy factors in OC explants exposed either to cisplatin or gentamicin.