Decrease of prestin expression by increased potassium concentration in organotypic cultures of the organ of Corti of newborn rats.

Prestin is the motor protein of the outer hair cells of the organ of Corti and a key factor in ensuring a high sensitivity level of mammalian hearing. In the present study, we examined the effects of increased extracellular potassium (K(+)) concentration on the expression of prestin mRNA and the transcription factors Gata-3 and Carf in the organotypic culture of the organ of Corti of newborn rats. Mannitol and NaCl were used to analyze possible effects of hyperosmotic stress or ion-specific changes, respectively. An increase in prestin expression by a factor of 1.5-2.0 was seen in cultures grown in the presence of 5mM K(+). Potassium concentration of 35 and 55 mM induced a parallel decrease in prestin and Carf expression, but Gata-3 expression increased. Mannitol had no effect on gene expression whereas increased NaCl concentrations decreased prestin, but not Carf expression. The data suggest that chronic depolarization might decrease the prestin expression and possibly contribute to hearing loss and tinnitus.

Expression analysis of prestin and selected transcription factors in newborn rats.

Transcription factors (TFs) have a central role to play in regulating gene expression. To analyze the co-expression patterns of selected TFs with the motor protein prestin of the outer hair cells, we applied an real-time PCR approach combining several kinds of information: (i) expression changes during postnatal development, (ii) expression changes by exposure of organotypic cultures of the organ of Corti to factors which significantly affect prestin expression [thyroid hormone (T4), retinoic acid (RA), butyric acid (BA), increased KCl concentration] and (iii) changes along the apical-basal gradient. We found that the mRNA levels of the TF Brn-3c (Pou4f3), a member of the POU family, are significantly associated with the regulation of prestin during postnatal development and in cultures supplemented with T4 (0.5 µM), BA (0.5-2.0 mM), and high KCl (50 mM) concentration. The mRNA level of the constitutively active TF C/ebpb (CCAAT/enhancer binding protein beta) correlates positively with the prestin expression during postnatal development and in cultures exposed to T4 and RA (50-100 µM). The mRNA levels of the calcium-dependent TF CaRF correlates significantly with the prestin expression in cultures exposed to T4 and high KCl concentration. The observed coexpression patterns may suggest that the TFs Brn-3c, C/ebpb, and Carf contribute to regulating the expression of prestin under the investigated conditions.

Effects of retinoic acid and butyric acid on the expression of prestin and Gata-3 in organotypic cultures of the organ of corti of newborn rats.

Prestin is the motor protein of the outer hair cells of the organ of Corti and a key factor in ensuring a high level of sensitivity of mammalian hearing. The factors that influence prestin expression are still largely unknown. We studied the effects of the application of retinoic acid, a ligand of a nuclear receptor, and of butyric acid, an inhibitor of histone deacetylase activity, on the expression of mRNA of prestin and Gata-3 in the organotypic culture of the organ of Corti of newborn rats using RT-PCR. Application of retinoic acid at concentrations of 1-50 µM results in a dose-dependent expression decrease after two days in culture. Treatment with sodium butyrate (0.5-2 mM) elevated the expression of prestin and Gata-3. Statistically significant correlations between Gata-3 and prestin mRNA levels were observed under all conditions. The data indicate that retinoid nuclear transcription factors, GATA-3 and histone acetylation/deacetylation processes may have a regulatory role to play in prestin expression.

Expression of prestin and Gata-3,-2,-1 mRNA in the rat organ of Corti during the postnatal period and in culture.

Based on observations that mutations of GATA-3 are responsible for the HDR-syndrome (hypoparathyroidism, deafness, renal defects) and that GATA-transcription factors have an important role to play in inner ear development, we hypothesized that these transcription factors may be involved in regulatory changes of prestin transcription. To prove this, we examined in parallel the expression of mRNA of prestin and Gata-3,-2 and Gata-1 in the organ of Corti during early postnatal development of rats and in organotypic cultures. Remarkable relations are observed between prestin and Gata-3,-2 expression in organ of Corti preparations in vivo and in vitro: (i) Gata-3,-2 expression display similar apical-basal gradients as prestin mRNA levels. (ii) The prestin expression increases between postnatal day two and postnatal day eight by a factor of about four in the apical and middle segments and by a factor of two in the basal part. Highly significant Pearson correlation coefficients were observed between Gata-3,-2 mRNA and prestin levels when the data were evaluated by regression analyses. (iii) Parallel changes of prestin mRNA and Gata-3,-2 mRNA levels were observed in response to thyroid hormone and to gemfibrozil application. These observations suggest a regulatory role played by the Gata-3,-2 transcription factors in prestin expression.