Thyroid hormone is a critical determinant for the regulation of the cochlear motor protein prestin

Abstract

The most impressive property of outer hair cells (OHCs) is their ability to change their length at high acoustic frequencies, thus providing the exquisite sensitivity and frequency-resolving capacity of the mammalian hearing organ. Prestin, a protein related to a sulfate/anion transport protein, recently has been identified and proposed as the OHC motor molecule. Homology searches of 1.5 kb of genomic DNA 5' of the coding region of the prestin gene allowed the identification of a thyroid hormone (TH) response element (TRE) in the first intron upstream of the prestin ATG codon. Prestin(TRE) bound TH receptors as a monomer or presumptive heterodimer and mediated a triiodothyronine-dependent transactivation of a heterologous promotor in response to triiodothyronine receptors alpha and beta. Retinoid X receptor-alpha had an additive effect. Expression of prestin mRNA and prestin protein was reduced strongly in the absence of TH. Although prestin protein typically was redistributed to the lateral membrane before the onset of hearing, an immature pattern of prestin protein distribution across the entire OHC membrane was noted in hypothyroid rats. The data suggest TH as a first transcriptional regulator of the motor protein prestin and as a direct or indirect modulator of subcellular prestin distribution.

Figure 1

Localization of prestin mRNA and protein in cross sections of rat cochleae at P7 and P12 using in situ hybridization and immunohistochemistry. Note the restricted localization of mRNA to the apical part of OHC, whereas prestin protein becomes redistributed from the entire OHC membrane (P7) to the lateral plasma membrane (P12). D, Deiters cell. (Bar, 20 μm.)

Figure 2

Position of TRE^Prest within the gene. (A) Nucleotide sequence of the rat prestin gene containing the first coding exon (in italics). The protein-coding region is underlined. The putative translation initiation codon (ATG) is referred to as +1. A TRE at position −416, referred to as TRE^Prest, is boxed. Another putative TRE (6DR3) is located at position −516. (B) 5′ rapid amplification of cDNA ends PCR generated from P12 cochlear mRNA detected a 188-bp sequence (indicated as putative exons I and II), which does not match the exon containing the protein-coding region (exon III). (C) Based on homologous human genomic sequence, a model was designed proposing the position of TRE^Prest within the exon-intron map of the prestin gene. Note its position downstream of exons I and II and upstream of exon III.

Figure 3

DNA binding of TRs to TRE^Prest analyzed by EMSA. (A) Cell extract from the cochlea at P8 shifts ³²P-labeled TRE^Prest oligomers (lane 2) to a similar position to that of control [³²P]DR-4 (lane 1). The interaction is reduced in the presence of an excess of unlabeled TRE^Prest competitor oligomers (lane 3) but not in the presence of unlabeled oligomers specific for transcription factor IID or Oct-2A (lanes 4 and 5, respectively ). (B) Recombinant (rc.) TRα shifts [³²P]DR-4, [³²P]TRE^Prest, and [³²P]TREmutA to only one or both positions consistent with monomer and homodimer binding, respectively. (C) HeLa cell extract shifts [³²P]TRE^Prest to positions that resembled monomer, homodimer, and heterodimer receptor interaction (lane 2, three arrowheads); the presumptive heterodimeric interaction is similar to that observed with [³²P]DR-4 (lane 1). An excess of unlabeled TRE^Prest reduces the bands in lane 3. Modifications introduced in the first or second half-site of TRE^Prest influence either monomer or homodimer and heterodimer binding in competition assays (see Results). A schematic representation of the modified oligomers used as unlabeled competitors is given in D.

Figure 4

TRE^Prest confers T3 responsiveness to a heterologous promoter. TRE^Prest was inserted upstream of the simian virus 40 promoter of a luciferase reporter plasmid, which in itself was not T3-responsive. HEK293 cells were cotransfected with this reporter plasmid together with TRα, TRβ, or RXRα expression vectors. Luciferase activity was determined after 48 h of incubation in the presence or absence of T3 and T3 + all-trans-retinoic acid (ATRA), respectively. Luciferase values were corrected for transfection efficiency and variations in total protein concentration. The data are the mean ± SD of luciferase activity obtained from at least three independent transfections, each measured in triplicate.

Figure 5

Expression of prestin mRNA in rat cochleae using in situ hybridization (A and C) and Northern blot (B). (A) At P6, P8, and P16, in hypothyroid animals (Hypo) prestin mRNA is reduced severely in comparison to controls (Control). (B) At P8, the 6.4-kb prestin mRNA band is reduced significantly in the absence of TH, whereas mRNA approaches nearly control levels toward P22. Blots were probed with glyceraldehyde-3-phosphate dehydrogenase to ensure similar loading of mRNA per lane. (C) Expression of prestin mRNA in rat cochleae at P3 in the presence (Control P3) and absence (Hypo P3) of TH. Twelve hours after injecting hypothyroid rats with T4, prestin mRNA is up-regulated (Hypo P3+T4). No mRNA was detected when correspondent sense probes were used (Control P3, sense). P, Pillar cell. (Bar, 20 μm.)

Figure 6

Prestin protein expression and distribution in rat cochleae using laser confocal microscopy (A) and immunohistochemistry (B and C). (A) In hypothyroid animals (Hypo) a significant reduction of prestin protein is observed. Note the aberrant prestin distribution in hypothyroid P8 and P16 sections (open arrows). (Bar, 20 μm.) (B) Cochlea sections of control and hypothyroid animals at P28 were costained for prestin and synaptophysin (Syn). Note the immature prestin distribution across the entire OHC membrane including the synaptic pole of the cell (Hypo P28, asterisks). The synaptophysin expression in hypothyroid animals is reduced in comparison to controls (open arrows). (Bar, 10 μm.) (C) Prestin protein distribution in control and hypothyroid animals is compared with hypothyroid animals treated with T4 after parallel staining of prestin (red), 4′,6-diamidino-2-phenylindole (DAPI)-stained cell nuclei (blue), and synaptophysin (green) at P14. (Bar, 10 μm.)