| Increasing evidence suggests that hair cells undergo apoptosis secondary to cisplatin and aminoglycoside exposure. Although growth factors, oxygen free radical scavengers and iron chelators can protect hair cells from ototoxins, the intracellular signaling events leading to hair cell apoptosis have yet to be fully elucidated. To identify possible intracellular mediators of ototoxicity, we treated basal-turn, neonatal, rat hair cells in vitro with several specific intracellular signaling inhibitors, prior to cisplatin or gentamicin exposure. The cell permeable caspase inhibitor, Z-VAD-FMK (150 muM), provided potent protection of hair cells from cisplatin and gentamicin. Since caspases play a critical role in apoptosis, this result supports the apoptotic nature of hair cell death in cisplatin and gentamicin ototoxicity. Histochemical labeling studies using the TUNEL method and anti-activated caspase 3 antibodies further support the apoptotic nature of this hair cell death. The general G-protein inhibitor, GDP-betaS (1 mM), provided potent protection from gentamicin and cisplatin, suggesting involvement of a G-protein in the intracellular pathway linking cisplatin and gentamicin exposure to hair cell apoptosis. ADP-betaS had minimal effect against gentamicin, thus the protection is specific to a G-binding site, rather than a general reaction to nucleotides. Azido-GTP32 photolabeling studies indicated that an approximately 21 kDa G-protein is activated in the organ of Corti after exposure to cisplatin or gentamicin. MALDI/MS analysis of this band matched its sequence with c-H-p21 Ras with a high degree of certainty. A Raf pulldown assay also indicated that a 21 kDa protein binds to Raf in the organ of Corti after gentamicin exposure. The farnesyl transferase inhibitors FTase-inhibitor-1 (50 muM) and FTI-277 (10 muM) provided strong protection against ototoxicity, suggesting the possibility that activation of N- or K-Ras is functionally involved in OHC apoptosis. In contrast, MEK inhibitors increase hair cell loss, indicating hair cells require MEK/ERK for survival. Downstream pathways through which Ras may contribute to hair cell apoptosis include modulation of BAD and JNK phosphorylation. |
