Induction of heat shock proteins by hyperthermia and noise overstimulation in hsf1 -/- mice

Abstract

Diverse cellular and environmental stresses can activate the heat shock response, an evolutionarily conserved mechanism to protect proteins from denaturation. Stressors activate heat shock transcription factor 1 (HSF1), which binds to heat shock elements in the genes for heat shock proteins, leading to rapid induction of these important molecular chaperones. Both heat and noise stress are known to activate the heat shock response in the cochlea and protect it from subsequent noise trauma. However, the contribution of HSF1 to induction of heat shock proteins following noise trauma has not been investigated at the molecular level. We evaluated the role of HSF1 in the cochlea following noise stress by examining induction of heat shock proteins in Hsf1 ( +/- ) control and Hsf1 ( -/- ) mice. Heat stress rapidly induced expression of Hsp25, Hsp47, Hsp70.1, Hsp70.3, Hsp84, Hsp86, and Hsp110 in the cochleae of wild-type and Hsf1 ( +/- ) mice, but not in Hsf1 ( -/- ) mice, confirming the essential role of HSF1 in mediating the heat shock response. Exposure to broadband noise (2-20 kHz) at 106 dB SPL for 2 h produced partial hearing loss. Maximal induction of heat shock proteins occurred 4 h after the noise. In comparison to heat stress, noise stress resulted in lower induced levels of Hsp25, Hsp70.1, Hsp70.3, Hsp86, and Hsp110 in Hsf1 ( +/- ) mice. Induction of these heat shock proteins was attenuated, but not completely eliminated, in Hsf1 ( -/- ) mice. These same noise exposure conditions induced genes for several immediate early transcription factors and maximum induction occurred earlier than for heat shock proteins. Thus, additional signaling pathways and transcriptional regulators that are activated by noise probably contribute to induction of heat shock proteins in the cochlea.

FIG. 1

Induction of genes for Hsp70 by heat stress in Hsf1^+/− and Hsf1^−/− mice. Mice (three mice per sample) were subjected to heat shock (HS, n = 4), a sham procedure (SM, n = 2), or no treatment (control, n = 3) prior to euthanasia. Hsp70.1 or Hsp70.3 mRNA levels were determined by qRT-PCR and normalized to S16 mRNA. White bars represent fold change in Hsf1^+/− mice and black bars in Hsf1^−/− mice. Bars represent SEM.

FIG. 2

Basal levels of HSP mRNAs in the cochlea of Hsf1^−/− vs. Hsf1^+/− mice. qRT-PCR was performed on total RNA from the cochleae of unstressed Hsf1^−/− (n = 9) and Hsf1^+/− mice (n = 7), from six independent experiments. Ratios indicate the HSP mRNA levels in Hsf1^−/− mice compared to their paired Hsf1^+/− mice within each experiment. Ratios were subjected to paired Student’s t test. Asterisks denote statistically significant differences: ***p < 0.001. Bars indicate SEM.

FIG. 3

Time course of HSP gene induction following noise stress. Hsf1^+/− mice were exposed to BBN (106 dB SPL, 2–20 kHz, 2 h). Mice (four per sample) were euthanized either immediately after the noise exposure (t = 0) or at 1, 2, 4, 9, 12, and 24 h following noise. Control mice were not subjected to the noise stress. Cochlear RNA was isolated and subjected to real-time qRT-PCR to determine relative gene expression. Fold change is the ratio of normalized transcript level in the noise-exposed group to the control group. For all HSPs tested, maximum induction occurred at 4 h after noise.

FIG. 4

Effect of noise intensity on induction of HSP genes. Hsf1^+/− mice (four mice per sample) were exposed to noise for 2 h and euthanized 4 h after cessation of noise. Cochlear RNA was subjected to qRT-PCR: 98 dB (n = 7), 106 dB (n = 9), and 120 dB (n = 6). Fold change is the ratio of values for noise-exposed mice relative to unexposed controls. Values at 98 dB were compared to control values by t test. Differences among the three noise intensities were evaluated by one-way ANOVA, followed by post hoc pairwise comparisons to determine which differences account for the result of the more inclusive test. The p values for 106 and 120 dB vs. 98 dB were 0.0011 and 0.0002, respectively, below the sequential Bonferroni criterion for table-wide significance of multiple comparisons. **p < 0.01; ***p < 0.001. Bars indicate SEM.

FIG. 5

Effect of noise exposure on auditory thresholds. Baseline ABRs were measured on Hsf1^+/− mice (n = 15) 1–3 days prior to noise exposure (106 dB SPL, 2–20 kHz BBN, 2 h). ABRs were repeated 14 days after noise exposure. This noise exposure condition resulted in a slight permanent threshold shift (average of 12 dB SPL hearing loss at 12 kHz and ~20 dB SPL loss at 24 kHz) at 2 weeks after noise overstimulation. Bars represent SEM.

FIG. 6

Induction of Hsp genes following noise in Hsf1^+/− and Hsf1^−/− mice. Hsf1^+/− or Hsf1^−/− mice were exposed to 106 dB SPL BBN for 2 h and euthanized 4 h after noise exposure (n = 9 for each genotype). Induction of Hsps was analyzed by qRT-PCR. Fold change was calculated as in Figure 1 and subjected to unpaired Student’s t test; *p < 0.05; **p < 0.01. Bars represent SEM.

FIG. 7

Time course of IEG induction following noise stress. RNA samples used for the time course experiment (see Fig. 3) were also assayed for several IEGs by qRT-PCR as in Figure 3. Most IEGs tested showed maximum induction at 1 h, although IEGs such as c-Fos and LIF showed a second peak of activity at 4 h.