ABSTRACT
Deletion of thyroid hormone receptor beta (TR beta), a ligand-dependent transcription factor encoded by the Thrb gene, causes deafness and thyroid hyperactivity in Thrb-null (Thrb(tm1/tm1)) mice and in a recessive form of the human syndrome of resistance to thyroid hormone. Here, we have determined that a targeted mutation (Thra(tm2)) in the related Thra gene, encoding thyroid hormone receptor alpha suppresses these phenotypes in mice. Thra encodes a TR alpha 1 receptor which is non-essential for hearing and a TR alpha 2 splice variant of unknown function that neither binds thyroid hormone nor transactivates. The Thra(tm2) mutation deletes TR alpha 2 and concomitantly causes overexpression of TR alpha 1 as a consequence of the exon structure of the gene. Thra(tm2/tm2) mice have normal auditory thresholds indicating that TR alpha 2 is dispensable for hearing, and have only marginally reduced thyroid activity. However, a potent function for the Thra(tm2) allele is revealed upon its introduction into Thrb(tm1/tm1) mice, where it suppresses the auditory and thyroid phenotypes caused by loss of TR beta. These findings reveal a novel modifying function for a Thra allele and suggest that increased expression of TR alpha 1 may substitute for the absence of TR beta. The TR isotypes generated by the distinct Thrb and Thra genes represent a small family of receptors that have diverged to mediate different physiological roles; however, the ability of changes in Thra expression to compensate for loss of Thrb indicates that many functions of these genes remain closely related.
Subject(s)
DNA-Binding Proteins/genetics , Deafness/physiopathology , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Thyroid Hormone/genetics , Thyroid Gland/physiopathology , Animals , Body Weight , Cochlea/cytology , Cochlea/metabolism , DNA-Binding Proteins/physiology , Deafness/genetics , Evoked Potentials, Auditory, Brain Stem/physiology , Gene Deletion , Gene Expression , Genotype , Hair Cells, Auditory, Inner/physiology , Membrane Potentials/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Mutant Strains , Mutation , Potassium Channels/physiology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Thyroid Hormone/physiology , Suppression, Genetic , Thyroid Gland/metabolism , Thyrotropin/blood , Thyroxine/blood , Triiodothyronine/bloodABSTRACT
Type 1 deiodinase (D1) metabolizes different forms of thyroid hormones to control levels of T3, the active ligand for thyroid hormone receptors (TR). The D1 gene is itself T3-inducible and here, the regulation of D1 expression by TRalpha1 and TRbeta, which act as T3-dependent transcription factors, was investigated in receptor-deficient mice. Liver and kidney D1 mRNA and activity levels were reduced in TRbeta(-/-) but not TRalpha1(-/-) mice. Liver D1 remained weakly T3 inducible in TRbeta(-/-) mice whereas induction was abolished in double mutant TRalpha1(-/-)TRbeta(-/-) mice. This indicates that TRbeta is primarily responsible for regulating D1 expression whereas TRalpha1 has only a minor role. In kidney, despite the expression of both TRalpha1 and TRbeta, regulation relied solely on TRbeta, thus revealing a marked tissue restriction in TR isotype utilization. Although TRbeta and TRalpha1 mediate similar functions in vitro, these results demonstrate differential roles in regulating D1 expression in vivo and suggest that tissue-specific factors and structural distinctions between TR isotypes contribute to functional specificity. Remarkably, there was an obligatory requirement for a TR, whether TRbeta or TRalpha1, for any detectable D1 expression in liver. This suggests a novel paradigm of gene regulation in which the TR sets both basal expression and the spectrum of induced states. Physiologically, these findings suggest a critical role for TRbeta in regulating the thyroid hormone status through D1-mediated metabolism.
Subject(s)
Iodide Peroxidase/metabolism , Kidney/enzymology , Liver/enzymology , Receptors, Thyroid Hormone/metabolism , Animals , Body Weight , Female , Gene Expression Regulation, Enzymologic , Hyperthyroidism/enzymology , Hyperthyroidism/genetics , Hypothyroidism/enzymology , Hypothyroidism/genetics , Iodide Peroxidase/genetics , Kidney/drug effects , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Mutant Strains , Organ Specificity , Receptors, Thyroid Hormone/deficiency , Receptors, Thyroid Hormone/genetics , Triiodothyronine/metabolism , Triiodothyronine/pharmacologyABSTRACT
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.
Subject(s)
Cochlea/enzymology , Gene Expression Regulation, Developmental , Hearing , Iodide Peroxidase/biosynthesis , Isoenzymes/biosynthesis , Nerve Tissue Proteins/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Cochlea/growth & development , Enzyme Induction , In Situ Hybridization , Iodide Peroxidase/genetics , Isoenzymes/genetics , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Thyroxine/metabolism , Triiodothyronine/biosynthesis , Iodothyronine Deiodinase Type IIABSTRACT
Melatonin was measured in a species of aerobic photosynthetic bacteria, Erythrobacter longus, grown in either constant light or constant dark. A radioimmunoassay was used to quantify melatonin levels and thin-layer chromatography to confirm the identity of melatonin immunoactivity. Melatonin levels were significantly higher (nearly 2.3-fold) in the dark-grown than in the light-grown samples. Also, the homogenates of the dark-grown bacteria retained melatonin-producing enzymatic activity, whereas the light-grown homogenates did not; melatonin levels extracted from the dark-grown homogenates increased with increasing extraction time, reaching as high as 29.2 ng.mg-1 protein at 120 min. Removal of membrane fragments from homogenates did not influence melatonin levels in light-grown homogenate, but this procedure increased melatonin levels in dark-grown homogenate, indicating that at least some of the enzymes in the pathway of melatonin production are not membrane-bound. This study is the second to demonstrate the presence of melatonin at the prokaryotic level, supporting the evidence that melatonin appeared very early in evolution. Its function in prokaryotes has not been determined, but may relate to its antioxidative actions.
