Divergent roles for thyroid hormone receptor beta isoforms in the endocrine axis and auditory system - PubMed (original) (raw)

Divergent roles for thyroid hormone receptor beta isoforms in the endocrine axis and auditory system

E D Abel et al. J Clin Invest. 1999 Aug.

Abstract

Thyroid hormone receptors (TRs) modulate various physiological functions in many organ systems. The TR alpha and TR beta isoforms are products of 2 distinct genes, and the beta 1 and beta 2 isoforms are splice variants of the same gene. Whereas TR alpha 1 and TR beta 1 are widely expressed, expression of the TR beta 2 isoform is mainly limited to the pituitary, triiodothyronine-responsive TRH neurons, the developing inner ear, and the retina. Mice with targeted disruption of the entire TR beta locus (TR beta-null) exhibit elevated thyroid hormone levels as a result of abnormal central regulation of thyrotropin, and also develop profound hearing loss. To clarify the contribution of the TR beta 2 isoform to the function of the endocrine and auditory systems in vivo, we have generated mice with targeted disruption of the TR beta 2 isoform. TR beta 2-null mice have preserved expression of the TR alpha and TR beta 1 isoforms. They develop a similar degree of central resistance to thyroid hormone as TR beta-null mice, indicating the important role of TR beta 2 in the regulation of the hypothalamic-pituitary-thyroid axis. Growth hormone gene expression is marginally reduced. In contrast, TR beta 2-null mice exhibit no evidence of hearing impairment, indicating that TR beta 1 and TR beta 2 subserve divergent roles in the regulation of auditory function.

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Figures

Figure 1

Targeted disruption of the TRβ2 exon. (a) Schematic representation of the TRβ2 exon and flanking DNA sequence. The targeting vector was generated by deleting a 1-kb _Nco_I-_Sac_I fragment encompassing the entire coding exon of the TRβ2 NH2-terminus and the adjacent intron, and by inserting the neomycin resistance gene under the influence of the phosphoglycerate kinase promoter. Homologous integration of the targeting vector was confirmed by Southern blot of genomic DNA digested with _Nco_I and probed with an α-32P–labeled, 700-bp _Eco_RI-_Bam_HI DNA fragment that is immediately upstream of the start of the targeting vector. The values 3.4 kb and 4.0 kb represent the expected sizes of the hybridized band in the WT and targeted alleles, respectively. B, _Bam_HI; E, _Eco_RI; N, _Nco_I; S, _Sac_I. (b) Representative Southern blot, demonstrating the targeted and WT alleles in KO (–/–), heterozygous (+/–), and WT (+/+) mice.

Figure 2

TR isoform expression. (a) RT-PCR of pituitary RNA (pooled from 6 KO and 6 WT mice), demonstrating the absence of the TRβ2 transcript in KO mice. The 5′ PCR primers used were specific for TRβ2 and TRβ1 (denoted A and B, respectively). The 3′ primer (denoted C) was common to both TR isoforms. L, molecular weight ladder; WT, wild-type; KO, knockout; ø, no added RNA. (b) RNase protection assays examining TRβ and TRα expression in multiple tissues of WT and KO mice. Br, brain; Ht, heart; Lu, lung; Li, liver; Ki, kidney.

Figure 3

Basal serum concentrations of T4, T3, and TSH in KO and WT mice. Numbers of animals are 13 KO and 18 WT for T4 assays, 18 KO and 14 WT for TSH assays, and 8 KO and 8 WT for T3 assays. ***P < 0.0001, **P < 0.01, and *P < 0.02 KO vs. WT.

Figure 4

TSH and T4 response to T3 administration. (a) Northern blots of pituitary RNA from WT and KO mice at baseline (0) and after 3 weeks of T3 administration (3). Blots were probed for TSHβ and cyclophillin. Note the complete suppression of TSHβ message by T3 in WT mice, and the partial suppression in KO mice. (b) Densitometric analysis of TSHβ Northern blots corrected for loading with cyclophillin densities. *WT TSHβ mRNA after T3 suppression was not detectable above background counts. (c) Serum T4 measured at weekly intervals in KO (n = 8) and WT (n = 6) mice. Mice were injected daily with T3 for the entire 3-week period. *P < 0.02, **P < 0.01, ***P < 0.001 vs. WT.

Figure 5

TSH response to hypothyroidism. (a) Northern blot of total RNA obtained from pooled pituitaries (n = 5 for WT and KO), demonstrating TSHβ mRNA responses in hypothyroid WT and KO mice. Amount of RNA loaded in each lane (in micrograms) is shown. (b) Densitometry of TSHβ mRNA abundance corrected for loading by hybridizing the same membranes with a cDNA for actin. Note the reduced response to hypothyroidism in KO vs. WT mice.

Figure 6

GH gene expression. (a) Representative Northern blot of total RNA obtained from pooled pituitaries for WT (first 2 lanes) and KO (last 2 lanes), demonstrating GH mRNA at ambient (basal) T4 concentrations and after administration of high doses of T3. Blots were also probed for cyclophillin. Five micrograms of RNA is loaded in each lane. Each lane represents RNA extracted from 3 pituitary glands. (b) Densitometry of GH mRNA abundance corrected for loading by hybridizing the same membranes with a cDNA for cyclophillin. Note the reduced basal GH expression and the blunted response to T3.

Figure 7

Evidence that cochlear function is normal in TRβ2 KO mice. ABRs in 8-week-old KO (n = 4) and WT (n = 3) mice. Threshold SPLs (0.0002 dyn/cm2) required for the detection of an ABR were obtained across a range of test-tone frequencies. Note that at all frequencies tested, TRβ2 KO mice exhibit no abnormality in ABR thresholds.

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Divergent roles for thyroid hormone receptor beta isoforms in the endocrine axis and auditory system - PubMed (original) (raw)