Thyroid hormone signaling during a postnatal period in the mouse is essential for cochlear development and the subsequent onset of hearing. To study the control of this temporal dependency, we investigated the role of iodothyronine deiodinases, which in target tissues convert the prohormone thyroxine into triiodothyronine (T3), the active ligand for the thyroid hormone receptor (TR). Type 2 5 -deiodinase (D2) activity rose dramatically in the mouse cochlea to peak around postnatal day 7 (P7), after which activity declined by P10. This activity peak a few days before the onset of hearing suggests a role for D2 in amplifying local T3 levels at a critical stage of cochlear development. A mouse cochlear D2 cDNA was isolated and demonstrated near identity to rat D2. In situ hybridization localized D2 mRNA in periosteal connective tissue in the modiolus, the cochlear outer capsule and the septal divisions between the turns of the cochlea. Surprisingly, D2 expression in these regions that give rise to the bony labyrinth was complementary to TR expression in the sensory epithelium. Thus, the connective tissue may control deiodination of thyroxine and release of T3 to confer a paracrine-like control of TR activation. These results suggest that temporal and spatial control of ligand availability conferred by D2 provides an unexpectedly important level of regulation of the TR pathways required for cochlear maturation.development ͉ thyroid hormone receptor ͉ deiodinase ͉ cochlea O ne of the most sensitive functions controlled by thyroid hormone (TH) is the development of hearing, as is evident from the deafness associated with human congenital hypothyroidism. Experimental hypothyroidism in rodents has revealed that the cochlea is a major site of TH action and has shown that cochlear maturation and the onset of auditory function around postnatal day 14 (P14) require TH during a critical period between the late embryonic stage and the second postnatal week (1-3). This developmental window is strictly delineated, because TH can rescue cochlear morphogenesis and auditory function in hypothyroid mice or rats if provided during early postnatal stages but not after P10-P12. Little is known of the mechanisms that control this temporal regulation.TH receptors (TR) play a critical role in the auditory system (4). TR␣1 and TR␤, encoded by two related genes, are nuclear receptors that act as ligand-dependent transcription factors (5, 6). TR␣1 and TR␤ are expressed in the cochlea with TR␤ mRNA being prominent both in the embryonic otic vesicle and in the postnatal sensory epithelium and spiral ganglion (7-9). TR␤-deficient mice and humans are severely deaf, as assessed by defective auditory-evoked brainstem responses (10, 11), whereas TR␣1-deficient mice have a normal auditory-evoked brainstem response (12). Mild hearing defects occur in Ϸ20% of cases of the human syndrome of resistance to thyroid hormone, which is associated with TR␤ point mutations (13).The control of ligand availability in target tissues constitutes another level of ...

Three "P-box" amino acids within the DNA recognition alpha-helix of members of the steroid hormone and thyroid hormone families of nuclear receptors are known to determine the identity of two of the six base pairs within the half-sites of cognate DNA elements. We introduced P-box substitutions derived from different members of the thyroid hormone/estrogen receptor (T3R/ER) family into the beta-isoform of human thyroid hormone receptor (hT3R beta) and tested the DNA binding and transactivation activities of these mutants using thyroid hormone response elements (TREs) with half-sites composed of different sequences and arranged in different orientations. Different P-box sequences derived from the T3R/ER family resulted in distinct DNA binding specificities determined by the fourth base pair of the half-site. Thyroid hormone receptor mutants containing EGA, EAA, EGS substitutions for the wild type EGG P-box bound with wild type affinity to consensus AGGTCA half-sites, regardless of orientation. TREs composed of AGGACA half-sites bound hT3R beta s with an EGG or EAA P-box sequence, but not those with EGA or EGS P-box sequence. A reversal of this specificity was observed on a direct repeat TRE with AGGGCA half-sites. Additionally, an ESG P-box substitution in hT3R beta prevented the receptor from binding to a direct repeat as a homodimer, but this mutant could bind as a heterodimer with retinoid X receptor or to the everted repeat TRE from the chicken lysozyme promoter.

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Nuclear hormone receptors bind to hormone response elements in DNA consisting of two half-sites of 6 base pairs. The P-box amino acids of each receptor determine the identities of the central nucleotides of the half-site. 57 P-box variants of the human thyroid hormone receptor (hT3R␤) were used to demonstrate the relationship between P-box sequence and DNA binding specificity by homodimers and heterodimers formed with the retinoid X receptor (RXR). In general, the formation of heterodimers relieved many of the constraints on the compatibility of hT3R␤ P-box sequences with DNA binding. Effects were most dramatic for heterodimers bound to a direct repeat spaced by four base pairs. RXR also overrides the P-box-derived DNA binding specificity of hT3R␤ when heterodimers are bound to inverted or everted repeat elements. These effects of RXR are most pronounced on AGGTCA half-sites but are squelched when the RXR partner of the heterodimer is bound to an AGGACA half-site. The influence of RXR on hT3R␤ DNA binding specificity varies with the orientation of halfsites in the element, the identity of the fourth base pair of the half-site, and the spacing between the half-sites of direct repeats. These differences suggest that the DNA binding domains of RXR-hT3R␤ heterodimers are not positioned equivalently on the various elements, affecting the manner in which the P-box amino acids of hT3R␤ interact with base pairs within the half-site.The thyroid hormone receptor (T3R) 1 is a ligand-responsive transcription factor within the superfamily of nuclear receptors that includes the steroid, retinoid, and vitamin D 3 receptors (1-3). In general, receptors in this superfamily mediate their transcriptional responses through highly conserved DNA binding domains (DBDs), which recognize half-site motifs of AGNNCA (4 -6). Structural analyses of several nuclear receptor DBDs have shown that this domain is composed of two zinc finger-like modules, each followed by an amphipathic ␣-helix (see Fig. 1A) (4, 5, 7). The ␣-helix following the first finger motif is the DNA recognition ␣-helix and is positioned in the major groove of the DNA. Within the DNA recognition ␣-helix, a conserved lysine and a conserved arginine residue make contacts to the conserved guanines of the half-site. The ␣-helix following the second finger motif folds perpendicularly over the DNA recognition ␣-helix, stabilizing the structure and providing phosphate contacts to the DNA. In the case of the T3R DBD, an additional ␣-helix downstream of the zinc fingers provides additional phosphate and minor groove interactions with the DNA (7) .Discrimination of the central base pairs of the hexameric half-site is determined largely by the identity of three amino acids in the DNA recognition ␣-helix, referred to as the P-box (see Fig. 1) (8 -10). Nuclear receptors have been divided into two subfamilies according to the identity of their P-box amino acid sequences (2, 11). The subfamily of receptors containing a GSV P-box sequence optimally recognize DNA elements containing AGAACA...