Celastrol inhibits aminoglycoside-induced ototoxicity via heat shock protein 32

Abstract

Hearing loss is often caused by death of the mechanosensory hair cells of the inner ear. Hair cells are susceptible to death caused by aging, noise trauma, and ototoxic drugs, including the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Ototoxic drugs result in permanent hearing loss for over 500,000 Americans annually. We showed previously that induction of heat shock proteins (HSPs) inhibits both aminoglycoside- and cisplatin-induced hair cell death in whole-organ cultures of utricles from adult mice. In order to begin to translate these findings into a clinical therapy aimed at inhibiting ototoxic drug-induced hearing loss, we have now examined a pharmacological HSP inducer, celastrol. Celastrol induced upregulation of HSPs in utricles, and it provided significant protection against aminoglycoside-induced hair cell death in vitro and in vivo. Moreover, celastrol inhibited hearing loss in mice receiving systemic aminoglycoside treatment. Our data indicate that the major heat shock transcription factor HSF-1 is not required for celastrol-mediated protection. HSP32 (also called heme oxygenase-1, HO-1) is the primary mediator of the protective effect of celastrol. HSP32/HO-1 inhibits pro-apoptotic c-Jun N-terminal kinase (JNK) activation and hair cell death. Taken together, our data indicate that celastrol inhibits aminoglycoside ototoxicity via HSP32/HO-1 induction.

Figure 1

Celastrol induces HSPs and inhibits aminoglycoside-induced hair cell death. (a) RT-PCR analysis of HSP mRNA expression. Utricles were treated with (or without) celastrol for 3 h and allowed to recover for 0–11 h. Celastrol resulted in robust upregulation of HSP70 and HSP32/HO-1 mRNA levels relative to control utricles. More modest upregulation of HSP90 and HSP27 mRNA were observed with celastrol. Error bars represent S.E.M. on real-time qRT-PCRs performed in triplicate. (b) Western blot analysis of celastrol-mediated HSP induction. Utricles treated with a 43°C heat shock were used for comparison. Celastrol induced HSP70 expression levels that were comparable to those induced by heat shock. Celastrol-mediated HSP32 induction was greater than that induced by heat shock. (c and g) Celastrol inhibits aminoglycoside-induced hair cell death. (c) Control utricles display normal hair cell densities. (d) Celastrol does not decrease hair cell viability. (e) Neomycin results in a significant loss of hair cells. (f) Celastrol inhibits neomycin-induced hair cell death. (g) Celastrol inhibits neomycin-induced hair cell death across the dose–response relationship (2-way ANOVA F_1,132=28.916, P<0.001). (h) Celastrol inhibits aminoglycoside-induced JNK activation. JNK activation was analyzed by phosphorylation status. Celastrol (1.5 μM for 3 h) does not result in JNK activation. Neomycin resulted in robust JNK activation relative to basal levels. Neomycin-induced JNK activation is inhibited by celastrol. Scale bar in f=20 μM. Asterisks in g denote a significant difference in neomycin-induced hair cell death between celastrol-treated and untreated utricles by post hoc tests

Figure 2

Hsf-1 is not required for celastrol-mediated protection or HSP32/HO-1 induction. (a) Utricles from Hsf-1^+/+ and Hsf-1^−/− mice were treated with celastrol (1.5 μM for 3 h) and/or gentamicin (5 mM for 24 h). Celastrol did not result in loss of hair cells in utricles from either Hsf-1^+/+ or Hsf-1^−/− mice. Gentamicin resulted significant hair cell loss in utricles of both genotypes. Celastrol inhibited gentamicin-induced hair cell death in both Hsf-1^+/+ and Hsf-1^−/− utricles (3-way ANOVA F_1,59=34.24, P<0.001). (b) Celastrol induces HSP32/HO-1 expression in the absence of Hsf-1. Utricles from Hsf-1^−/− mice and their wild-type littermates were incubated in celastrol for 3 h before RNA extraction. Celastrol resulted in ∼100-fold increase in HSP70 mRNA expression in utricles from Hsf-1^+/+ mice, but it did not result in increased HSP70 mRNA in utricles from Hsf-1^−/− mice. In contrast, celastrol treatment resulted in similar increases in HSP32/HO-1 mRNA levels in utricles of both Hsf-1^+/+ and Hsf-1^−/− mice. Error bars represent S.E.M. on real-time qRT-PCRs performed in triplicate

Figure 3

Celastrol induces Nrf2 activation. (a) RT-PCR analysis of Nrf2 mRNA levels in response to celastrol. Nrf2 mRNA expression peaked after 3 h of celastrol incubation and declined thereafter. Error bars represent S.E.M. on real-time qRT-PCRs performed in triplicate. (b–g) Nrf2 immunolocalization. Hair cell nuclei were labeled with Hoescht. (b–d) In control utricles Nrf2 is localized to the hair cell cytosol, and the nuclei appear as dark voids (arrows in c). (e–g) Celastrol results in increased Nrf2 immunoreactivity as well as Nrf2 translocation to the nucleus. Scale bar in g=20 μM. (h) qRT-PCR analysis of Nrf2 transcriptional targets. GST, NQO1, UDP, and GGC expression were all increased in celastrol-treated utricles relative to control levels. Error bars represent S.E.M. on real-time qRT-PCRs performed in triplicate

Figure 4

HSP32/HO-1 is the major mediator of celastrol's protective effect. (a) HSP32/HO-1 is required for the protective effect of celastrol. Utricles from both HSP70^−/− and HSP70^+/+ mice were treated with celastrol and the HSP32/HO-1 inhibitor ZnPPIX. Gentamicin resulted in significant hair cell death in utricles of both genotypes. Celastrol inhibited gentamicin-induced hair cell death in both HSP70^−/− and HSP70^+/+ utricles. ZnPPIX abolished the protective effect of celastrol in both HSP70^−/− and HSP70^+/+ utricles (1-way ANOVA F_1,29=26.35, P<0.0001). Asterisks indicate significance (P<0.05) by post hoc analysis. (b) HSP32/HO-1 inhibits aminoglycoside-induced hair cell death. Utricles were treated with the HSP32/HO-1 inducer CoPPIX. HSP32/HO-1 induction inhibited aminoglycoside-induced hair cell death (2-way ANOVA F_1,9=10.36, P<0.011). (c) HSP32/HO-1 inhibits aminoglycoside-induced JNK activation. Neomycin increased JNK activation, and this activation was inhibited by CoPPIX. (d) CO inhibits aminoglycoside-induced hair cell death. Utricles were treated with CORM2, a CO-releasing molecule. CORM2 partially inhibited neomycin-induced hair cell death (1-way ANOVA F_1,25=79.29, P<0.0001)

Figure 5

Celastrol inhibits aminoglycoside-induced hearing loss and hair cell death in vivo. Top panel: hearing testing via ABR analysis was used to determine whether celastrol inhibits aminoglycoside-induced hearing loss in vivo. Pre-test ABR thresholds were determined, then mice received daily injections of celastrol and/or kanamycin for 14 days. ABR hearing thresholds were again measured 3 weeks after the final injections, and data are reported as threshold shifts (that is, the difference between pre-test and post-test thresholds). Mice treated with kanamycin showed significant hearing losses (threshold shifts) at all frequencies tested. Mice treated with celastrol plus kanamycin had significantly smaller threshold shifts than mice given kanamycin alone (RM-ANOVA, F_3,12=104.77, P<0.0001, n=16). Asterisks indicate significance (P<0.05) by post hoc analysis. Lower panel: celastrol inhibits aminoglycoside-induced cochlear hair cell death in vivo. Myosin VIIa immunohistochemistry was used to label hair cells in cochleas of mice treated with kanamycin and celastrol. The normal mouse cochlea (shown for reference) contains a single row of inner hair cells (IHC) and three rows of outer hair cells (OHC 1–3). (a–c) The cochlea of a mouse treated with kanamycin shows some loss of outer hair cells in the apex (a) and total loss of outer hair cells in the middle (b) and base (c). Occasional loss of inner hair cells is also evident (b). (d and e) The cochlea of a mouse treated with kanamycin plus celastrol retains of the majority of outer hair cells in the apical and middle turns, and all inner hair cells are present. (f) Celastrol did not protect outer hair cells in the basal turn of the cochlea. Scale bar=30 μm

Figure 6

Potential mechanisms of celastrol-mediated protection against aminoglycoside-induced hair cell death. Aminoglycoside antibiotics result in increased ROS production and pro-apoptotic JNK activation in hair cells. Celastrol activates the Nrf2-transcription factor and induces HSP32/HO-1. HSP32/HO-1 inhibits pro-apoptotic JNK activation and hair cell death, possibly through the action of CO