Cisplatin-induced hair cell loss in zebrafish neuromasts is accompanied by protein nitration and Lmo4 degradation.

Generation of reactive oxygen species, a critical factor in cisplatin-induced ototoxicity, leads to the formation of peroxynitrite, which in turn results in the nitration of susceptible proteins. Previous studies indicated that LMO4, a transcriptional regulator, is the most abundantly nitrated cochlear protein after cisplatin treatment and that LMO4 nitration facilitates ototoxicity in rodents. However, the role of this mechanism in regulating cisplatin-induced hair cell loss in non-mammalian models is unknown. As the mechanosensory hair cells in the neuromasts of zebrafish share many features with mammalian inner ear and is a good model for studying ototoxicity, we hypothesized that cisplatin treatment induces protein nitration and Lmo4 degradation in zebrafish hair cells, thereby facilitating hair cell loss. Immunostaining with anti-parvalbumin revealed a significant decrease in the number of hair cells in the neuromast of cisplatin treated larvae. In addition, cisplatin treatment induced a significant decrease in the expression of Lmo4 protein and a significant increase in nitrotyrosine levels, in the hair cells. The cisplatin-induced changes in Lmo4 and nitrotyrosine levels strongly correlated with hair cell loss, implying a potential link. Furthermore, a significant increase in the expression of activated Caspase-3 in zebrafish hair cells, post cisplatin treatment, suggested that cisplatin-induced decrease in Lmo4 levels is accompanied by apoptosis. These findings suggest that nitrative stress and Lmo4 degradation are important factors in cisplatin-induced hair cell loss in zebrafish neuromasts and that zebrafish could be used as a model to screen the otoprotective efficacy of compounds that inhibit protein nitration.

Inhibition of protein nitration prevents cisplatin-induced inactivation of STAT3 and promotes anti-apoptotic signaling in organ of Corti cells.

JAK/STAT pathway is one among the several oxidative stress-responsive signaling pathways that play a critical role in facilitating cisplatin-induced ototoxicity. Cisplatin treatment decreases the levels of cochlear LMO4, which acts as a scaffold for IL6-GP130 protein complex. Cisplatin-induced nitration and degradation of LMO4 could destabilize this protein complex, which in turn could compromise the downstream STAT3-mediated cellular defense mechanism. Here, we investigated the link between cisplatin-induced nitrative stress and STAT3-mediated apoptosis by using organ of Corti cell cultures. SRI110, a peroxynitrite decomposition catalyst that prevented cisplatin-induced decrease in LMO4 levels and ototoxicity, was used to inhibit nitrative stress. Immunoblotting and immunostaining indicated that cisplatin treatment decreased the expression levels, phosphorylation, and nuclear localization of STAT3 in UB/OC1 cells. Inhibition of nitration by SRI110 co-treatment prevented cisplatin-induced inactivation of STAT3 and promoted its nuclear localization. SRI110 co-treatment reversed the cisplatin-induced changes in the expression levels of Bcl2l1, Ccnd1, Jak2, Jak3, and Src and significantly attenuated the changes in the expression levels of Cdkn1a, Egfr, Fas, Il6st, Jak1, Stat3, and Tyk2. Collectively, these results suggest that the inhibition of cisplatin-induced nitration prevents the inactivation of STAT3, which in turn enables the transcription of anti-apoptotic genes and thereby helps to mitigate cisplatin-induced toxicity.