Hsp70 Inhibits Aminoglycoside-Induced Hair Cell Death and is Necessary for the Protective Effect of Heat Shock

Abstract

Sensory hair cells of the inner ear are sensitive to death from aging, noise trauma, and ototoxic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to aminoglycosides results in hair cell death that is mediated by specific apoptotic proteins, including cJun N-terminal kinase (JNK) and caspases. Induction of heat shock proteins (Hsps) is a highly conserved stress response that can inhibit JNK-and caspasedependent apoptosis in a variety of systems. We have previously shown that heat shock results in a robust upregulation of Hsps in the hair cells of the adult mouse utricle in vitro. In addition, heat shock results in significant inhibition of both cisplatin-and aminoglycoside-induced hair cell death. In our system, Hsp70 is the most strongly induced Hsp, which is upregulated over 250-fold at the level of mRNA 2 h after heat shock. Therefore, we have begun to examine the role of Hsp70 in mediating the protective effect of heat shock. To determine whether Hsp70 is necessary for the protective effect of heat shock against aminoglycoside-induced hair cell death, we utilized utricles from Hsp70.1/3 −/− mice. While heat shock inhibited gentamicin-induced hair cell death in wild-type utricles, utricles from Hsp70.1/3 −/− mice were not protected. In addition, we have examined the role of the major heat shock transcription factor, Hsf1, in mediating the protective effect of heat shock. Utricles from Hsf1 −/− mice and wild-type littermates were exposed to heat shock followed by gentamicin. The protective effect of heat shock on aminoglycoside-induced hair cell death was only observed in wildtype mice and not in Hsf1 −/− mice. To determine whether Hsp70 is sufficient to protect hair cells, we have utilized transgenic mice that constitutively overexpress Hsp70. Utricles from Hsp70-overexpressing mice and wild-type littermates were cultured in the presence of varying neomycin concentrations for 24 h. The Hsp70-overexpressing utricles were significantly protected against neomycin-induced hair cell death at moderate to high doses of neomycin. This protective effect was achieved without a heat shock. Taken together, these data indicate that Hsp70 and Hsf1 are each necessary for the protective effect of heat shock against aminoglycoside-induced death. Furthermore, overexpression of Hsp70 alone significantly inhibits aminoglycoside-induced hair cell death.

Bax, Bcl2, and p53 Differentially Regulate Neomycin- and Gentamicin-Induced Hair Cell Death in the Zebrafish Lateral Line

“…For example, PFTα can inhibit zebrafish p73 in vivo and mammalian cyclin D1 in vitro, while PFTμ inhibits hsp70 and can interfere with autophagy (Davidson et al 2008;Leu et al 2009;Sohn et al 2009;Pimkina and Murphy 2011). Hsp70 activation inhibits aminoglycoside-induced hair cell loss in mammalian models, making it unlikely that inhibiting hsp70 would exert a protective effect in zebrafish hair cells (Taleb et al 2008(Taleb et al , 2009). However, blocking autophagy does provide a modest protective effect from aminoglycoside damage in zebrafish (Coffin et al 2013), so we cannot rule out this off-target effect of PFTμ in our present study, nor can we be certain that PFTα or PFTμ are not exerting an influence on additional targets.…”

Heat Shock Protein-Mediated Protection Against Cisplatin-Induced Hair Cell Death

“…HSP70 is required for the protective effect of heat shock against aminoglycoside-induced hair cell death (Taleb et al 2008;May et al 2013). In order to determine if HSP70 is also necessary for the protective effect of heat shock against cisplatin-induced hair cell death, we used utricles from HSP70.1/3 −/− and wildtype mice.…”

“…In order to determine if HSP70 is sufficient to protect hair cells against cisplatin-induced death, we utilized utricles from mice that constitutively express ratinducible HSP70 (rHSP70i CE) (Marber et al 1995;Kim et al 2006b;Taleb et al 2008Taleb et al , 2009). We first examined HSP induction in response to heat shock in utricles from rHSP70i CE mice and their wild-type littermates (Fig.…”

“…HSP70 inhibits multiple pro-apoptotic signals, including oligomerization of Bax, release of both cytochrome c and second mitochondria-derived activator of caspases (Smac) from mitochondria, formation of a functional apoptosome, and even cell death subsequent to caspase 3 activation (Jaattela et al 1998;Beere et al 2000;Tsuchiya et al 2003;Stankiewicz et al 2005;Jiang et al 2009;Evans et al 2010). Moreover, HSP70 inhibits aminoglycosideinduced hair cell death in vitro and hearing loss in vivo (Taleb et al 2008(Taleb et al , 2009May et al 2013). Our previous data indicate that HSP70 is the most highly induced HSP in response to heat shock in whole-organ cultures of utricles from mature mice (Cunningham and Brandon 2006;Taleb et al 2008;Francis et al 2011;May et al 2013).…”

“…Moreover, HSP70 inhibits aminoglycosideinduced hair cell death in vitro and hearing loss in vivo (Taleb et al 2008(Taleb et al , 2009May et al 2013). Our previous data indicate that HSP70 is the most highly induced HSP in response to heat shock in whole-organ cultures of utricles from mature mice (Cunningham and Brandon 2006;Taleb et al 2008;Francis et al 2011;May et al 2013). Our more recent studies indicate that supporting cells mediate the protective effect of HSP70 induction by secreting HSP70 (May et al 2013).…”