Involvement of ras activation in toxic hair cell damage of the mammalian cochlea

Abstract

To identify possible intracellular mediators of hair cell (HC) death due to ototoxins, we treated basal-turn, neonatal, rat HCs in vitro with several intracellular signaling inhibitors, prior to and during gentamicin exposure. The general guanine nucleotide-binding protein (G-protein) inhibitor, GDP-betaS (1 mM), provided potent HC protection, suggesting involvement of G-proteins in the intracellular pathway linking gentamicin exposure to HC death. ADP-betaS had minimal effect, indicating that the protection is specific to guanosine diphosphate (GDP)-binding, rather than a general reaction to nucleotides. Azido-GTP(32) photolabeling and gel electrophoresis indicated activation of an approximately 21 kDa G-protein in HCs after exposure to gentamicin. Spectroscopic analysis of peptide fragments from this band matched its sequence with H-Ras. The Ras inhibitors B581 (50 microM) and FTI-277 (10 microM) provided potent protection against damage and reduced c-Jun activation in HC nuclei, suggesting that activation of Ras is functionally involved in damage to these cells due to gentamicin. INVOLVEMENT OF RAS ACTIVATION IN TOXIC HAIR CELL DAMAGE

A PI3K Pathway Mediates Hair Cell Survival and Opposes Gentamicin Toxicity in Neonatal Rat Organ of Corti

“…Gentamicin-induced damage to hair cells that we observed in culture is similar to that reported previously (Battaglia et al 2003;Bodmer et al 2002a). Moreover, the agreement we observed between phalloidin and MyoVIIa labeling suggests that we did not miss surviving Btopless^hair cell somata in our assays.…”

“…This observation is consistent with current concepts of apoptosis regulation in cells, according to which cells are thought to exist in a finely tuned balance between survival and cell death (Raff 1992;Boatright and Salvesen 2003). Exposure of cells to stress disrupts this balance, and can activate opposing survival-and death-promoting pathways.…”

“…Other recent evidence suggests that the intracellular signals leading to JNK activation in hair cells include the small G proteins Ras and Rac/cdc42 (Bodmer et al 2002a). In particular, K-Ras may activate the pathway leading to JNK phosphorylation (Battaglia et al 2003). Other pathways that promote cell death in ototoxicity have also been identified (e.g., Wu et al 2002;McFadden et al 2003).…”

“…In addition to pathways that promote hair cell death, gentamicin exposure also appears to activate opposing pathways that promote hair cell survival (Battaglia et al 2003). This observation is consistent with current concepts of apoptosis regulation in cells, according to which cells are thought to exist in a finely tuned balance between survival and cell death (Raff 1992;Boatright and Salvesen 2003).…”

“…Treatment with low concentrations of the Ras inhibitor, FTI-277, sufficient to block the activation of HRas, enhanced gentamicin-induced hair cell death, whereas concentrations that were high enough to block K-Ras protected hair cells (Battaglia et al 2003). This observation is consistent with the activation of two signal transduction pathways in hair cells: an HRas/Raf/MEK/Erk pathway promoting hair cell survival, and a K-Ras/Rac/JNK pathway leading to hair cell death.…”