Inner ear supporting cells protect hair cells by secreting HSP70

Abstract

Mechanosensory hair cells are the receptor cells of hearing and balance. Hair cells are sensitive to death from exposure to therapeutic drugs with ototoxic side effects, including aminoglycoside antibiotics and cisplatin. We recently showed that the induction of heat shock protein 70 (HSP70) inhibits ototoxic drug-induced hair cell death. Here, we examined the mechanisms underlying the protective effect of HSP70. In response to heat shock, HSP70 was induced in glia-like supporting cells but not in hair cells. Adenovirus-mediated infection of supporting cells with Hsp70 inhibited hair cell death. Coculture with heat-shocked utricles protected nonheat-shocked utricles against hair cell death. When heat-shocked utricles from Hsp70-/- mice were used in cocultures, protection was abolished in both the heat-shocked utricles and the nonheat-shocked utricles. HSP70 was detected by ELISA in the media surrounding heat-shocked utricles, and depletion of HSP70 from the media abolished the protective effect of heat shock, suggesting that HSP70 is secreted by supporting cells. Together our data indicate that supporting cells mediate the protective effect of HSP70 against hair cell death, and they suggest a major role for supporting cells in determining the fate of hair cells exposed to stress.

Figure 1. Effects of heat shock on HSP levels.

(A) Control and heat-shocked utricles from CBA/J mice were examined for expression levels of HSPs using Western blotting. Heat shock results in upregulation of HSP70, HSP40, and HSP27. Numbers below each band indicate the fold change relative to the utricles that were not heat shocked. (B) Control and heat-shocked utricles from Hsp70^+/+ and Hsp70^–/– mice were examined for Hsp mRNA expression using real-time quantitative PCR (RT-qPCR). Heat shock resulted in a robust induction of Hsp70 mRNA in utricles from Hsp70^+/+ mice. In heat-shocked utricles from Hsp70^–/– mice, Hsp27 mRNA was induced, but Hsp70 mRNA was not.

Figure 2. Heat shock results in HSP70 upregulation in supporting cells.

Control and heat-shocked (43°C for 30 minutes) utricles were allowed to recover from heat shock for 6 hours and were sectioned (see Methods). Sections were labeled using antibodies against myosin 7a (red, hair cell marker) and HSP70 (green). Nuclei were labeled with Hoechst dye (blue). Confocal microscopy was performed using identical settings for laser power, gain, offset, and zoom. Shown are a control utricle (A) and a heat-shocked utricle (B–E). Control utricles (A) show very little HSP70 immunoreactivity. Heat-shocked utricles (B–E) show robust upregulation of HSP70 in supporting cells. C–E show a higher-magnification image of a heat-shocked utricle. (C) Myosin 7a labeling of a single hair cell. (D) HSP70 immunoreactivity is present in supporting cells but not in the hair cell. (E) Merged image. (F) Schematic showing locations of the hair cell (HC) and supporting cells (SC) in C–E. Scale bars: 10 μm (A and B); 5 μm (C–E). Images are representative of 3 experiments for a total of 10 to 12 utricles per condition.

Figure 3. Adenovirus specifically infects supporting cells.

Utricles were infected with Ad-RFP for 2 hours and then cultured in control media for an additional 24 hours and fixed. Hair cells and supporting cells were visualized using myosin 7a (blue) and SOX2 (green) immunochemistry, respectively. Schematic shows the structure of the utricular sensory epithelium and the locations of the hair cells and supporting cells. Locations of confocal (optical) sections shown in the upper and lower panels are indicated by lines and arrows in the schematic. Upper panels: in confocal images taken at the level of the hair cell nuclei, the Ad-RFP signal appears in the spaces between hair cells and does not overlap with the hair cell marker myosin 7a. Lower panels: in confocal sections at the level of the supporting cell nuclei, the Ad-RFP signal colocalizes with the supporting cell marker SOX2. Ad-RFP infection results in RFP expression in supporting cells only, and no hair cells are infected. Scale bar: 20 μm.

Figure 4. Adenoviral infection of supporting cells with HSP70 inhibits neomycin-induced hair cell death.

Utricles were infected with Ad-HSP70 (or Ad-GFP). Under these conditions, the adenovirus infects supporting cells, and no hair cells are infected (see Figure 3). Hair cells are labeled with myosin 7a. (A) Control utricles show normal hair cell numbers. (B) Infection with Ad-GFP does not result in hair cell loss (for clarity, the GFP channel is not shown). (C) Infection with Ad-HSP70 does not result in hair cell loss. (D) Neomycin treatment results in significant loss of hair cells. (E) Infection of supporting cells with Ad-GFP is not protective against neomycin-induced hair cell death. (F) Infection of supporting cells with Ad-HSP70 inhibits neomycin-induced hair cell death. (G) Western blot analysis of HSP70 expression in control and Ad-HSP70–infected utricles. (H) Ad-HSP70 is protective against neomycin-induced hair cell death. 1-way ANOVA, F_5,46 = 12.03, P < 0.0001. n = 5–12 utricles per condition. *P < 0.001 between neomycin and neomycin plus Ad-HSP70. Ctrl, control; Neo, neomycin.

Figure 5. Coculture with heat-shocked utricles is protective.

Utricles were cultured in 24-well plates containing Transwell permeable inserts. For each experiment, heat-shocked utricles were placed in the upper chamber of the Transwell device, and nonheat-shocked utricles were placed in the lower chamber. (A) Wild-type (Hsp70^+/+) utricles were cocultured. Control utricles and heat-shocked utricles had comparable hair cell densities. Gentamicin caused significant loss of hair cells. Heat-shocked utricles (upper chamber) were partially protected against gentamicin-induced hair cell death. Nonheat-shocked utricles (bottom chamber) were protected in a manner comparable to heat-shocked utricles. (B) When utricles from Hsp70^–/– mice were used in both chambers (heat-shocked and nonheat-shocked), protection against gentamicin-induced hair cell death was abolished in both groups of utricles. (C) Hsp70^–/– utricles were heat shocked and cultured in the upper chamber of the Transwell device, while Hsp70^+/+ utricles (nonheat shocked) were in the lower chamber. No protective effect against gentamicin-induced hair cell death was observed in either group. (D) Hsp70^+/+ utricles were heat shocked and cultured in the upper chamber of the Transwell device, while Hsp70^–/– utricles (nonheat shocked) were in the lower chamber. Both groups were protected against gentamicin-induced hair cell death. ANOVAs, *P < 0.05. n = 5–11 utricles per condition. Gent, gentamicin; HS, heat shocked.

Figure 6. HSP70 is secreted to the medium, and depletion of HSP70 from the medium abolishes the protective effect of coculture.

(A) HSP70 levels in medium from control and heat-shocked utricles were examined by ELISA. Medium surrounding heat-shocked utricles contained significantly more HSP70 than medium surrounding the control (nonheat-shocked) utricles (ANOVA, P < 0.0005). HSP70 levels in control tissue were comparable to those in control medium, while heat-shocked utricles contained significantly higher levels of HSP70. Shown are data from 3 biological replicates processed in parallel. The experiment was repeated 5 times with similar results. (B) Heat-shocked (upper chamber) and nonheat-shocked (lower chamber) utricles from Hsp70^+/+ mice were cocultured in a Transwell device containing 5 mM of gentamicin. As in Figure 5, gentamicin resulted in significant hair cell death, and both heat-shocked (upper) and nonheat-shocked (cocultured, lower) utricles were protected. Addition of a control IgG to the medium did not alter the protective effect of either heat shock (upper) or coculture with heat-shocked (lower) utricles. Depletion of HSP70 from the medium using an HSP70 function-blocking antibody (fbAb) abolished the protective effect of both heat-shocked (upper) and nonheat-shocked, cocultured (lower) utricles. ANOVA, *P < 0.05 relative to gentamicin. n = 5–15 utricles per condition.

Figure 7. Ad-HSP70-mCherry is protective in coculture.

Utricles were infected with Ad-HSP70-mCherry and placed in the upper chamber of a Transwell device containing 3 mM of neomycin. Noninfected utricles were placed in the lower chamber. No utricles were heat shocked. Infection of supporting cells with Ad-HSP70-mCherry resulted in significant inhibition of neomycin-induced hair cell death in both the infected utricles (upper) and noninfected utricles (lower). ANOVA, *P < 0.05 relative to neomycin alone. n = 6–11 utricles per condition.

Figure 8. Exogenous HSP70 is protective.

(A) Utricles were cultured in neomycin with (or without) recombinant human HSP70. Recombinant HSA was used as a control. Neomycin resulted in significant loss of hair cells (ANOVA, *P < 0.05 relative to control). Addition of exogenous HSP70 inhibited neomycin-induced hair cell death (ANOVA, ^#P < 0.05 relative to neomycin), while HSA was not protective. n = 5–20 utricles per condition. (B) Exogenous HSP70 is not internalized by hair cells. Neomycin-treated utricles were cultured in the presence of FITC-labeled recombinant human HSP70 (ExHSP70-FITC) or BSA (ExBSA-FITC) and examined for the presence of intracellular HSP70-FITC (or BSA-FITC) (green). Hair cells were labeled with an antibody against myosin 7a (red). Neither ExHSP70-FITC nor ExBSA-FITC was internalized by hair cells, although supporting cells internalized small amounts of both exogenous proteins. Scale bar: 10 μm (applies to all panels). Orth, orthogonal.

Figure 9. Ad-HSP70-mCherry is protective, and it is not internalized by hair cells.

(A) Supporting cells were infected with Ad-HSP70-mCherry (or Ad-mCherry as a control). Infected utricles were cultured in the presence or absence of neomycin (3 mM). Neither virus resulted in significant hair cell death, while neomycin caused a significant loss of hair cells. Infection of supporting cells with Ad-HSP70-mCherry inhibited neomycin-induced hair cell death (ANOVA, *P < 0.05). Infection of supporting cells with Ad-mCherry was not protective. n = 6–11 utricles per condition. (B) Utricles infected with Ad-HSP70-mCherry were fixed, and hair cells were labeled using antimyosin 7a (green). Utricles were examined using confocal microscopy (original magnification, ×63). Schematic shows the location of each panel. Upper panels show confocal images taken at the level of the hair cell cuticular plates, while the lower panels show confocal images from the same field taken at the level of the hair cell nuclei. Ad-HSP70-mCherry (red) is restricted to supporting cells and does not colocalize with myosin 7a (green, hair cells), indicating that Ad-HSP70-mCherry does not translocate from supporting cells to hair cells. Scale bars: 20 μm.