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Query: UNIPROT:P06889 (Mol)
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Thyroid hormone (T3) regulates the expression of rat TSH beta-subunit (TSH beta) mRNA, in part, at the posttranscriptional level, by reducing the half-life of TSH beta mRNA. The mechanism(s) mediating this alteration in mRNA stability are unknown, but previous work indicates that labile protein(s) are involved. The majority of cis-acting elements identified to date that have been implicated in the regulated destabilization of mRNAs have been located in the 3'-untranslated region (3'-UTR) of the mRNA. The 3'-UTR of rat, murine, and human TSH beta mRNA is highly conserved, and within this region is a 12-nucleotide consensus sequence, which is shared by the 3'-UTR of several other genes with unstable mRNAs. We reasoned that this homologous region could represent a binding motif for specific trans-acting RNA-binding protein(s), and that identification and characterization of such trans-acting factor(s) may provide critical insight into the mechanisms underlying T3-induced changes in TSH beta mRNA stability. Utilizing the RNA electrophoretic mobility shift assay and analysis of UV cross-linked RNA-protein complexes, a cytoplasmic trans-acting factor of approximately 80-85 kilodaltons was identified from rat pituitaries and several cell lines that binds in a sequence-specific manner to the 3'-UTR of rat TSH beta mRNA. Using competitive antisense oligonucleotides, the predominant binding site was mapped to the first 41 nucleotides of the 3'-UTR, which includes the consensus region. However, sequence upstream of the consensus was also shown to be important for binding. Using RNA electrophoretic mobility shift assay, two mRNAs containing sequence homology with the consensus region, c-erbA alpha-2 and a rat ferritin pseudogene, were shown to specifically compete with rat TSH beta mRNA for binding of this factor. Remarkably, the binding activity of this factor was regulated positively by T3 within 4 h, but only with rat pituitary extracts. These data suggest that in addition to binding rat TSH beta mRNA in a sequence-specific and T3-regulated manner, this novel trans-acting RNA-binding protein may also bind to other cytoplasmic mRNAs involved in diverse intracellular processes.
Mol Endocrinol 1995 Mar
PMID:Regulated specific protein binding to a conserved region of the 3'-untranslated region of thyrotropin beta-subunit mRNA. 777 83

Thyroid hormone action is not only determined by hormone availability, but also by target organ sensitivity. A dominant negative interaction is known to occur between thyroid hormone receptors (TRs) and the non-ligand binding splicing variant c-erbA alpha 2 as well as mutant TR beta 1 from kindreds with resistance to thyroid hormone. We compared the inhibitory effect of naturally occurring mutant hTR beta 1, artificially created hTR alpha 1 mutants, c-erbA alpha 2 and the human peroxisome proliferator-activated receptor (hPPAR) on three prototypic T3-response elements (TREs), TRE-PAL, DR + 4 and TRE-LAP. The inhibitory effect of mutant hTR alpha 1 and beta 1 occurred only on TRE-LAP and to a minor degree on DR + 4 when equimolar ratios of mutant/wildtype receptor were present. In contrast, the c-erbA alpha 2 splicing variant and the hPPAR inhibited TR action on all three TREs. Gel mobility shift experiments in the presence of T3 showed increased binding of mutant hTR alpha 1 and beta 1 only to TRE-LAP compared to the binding of wildtype hTRs, thereby explaining their TRE-selective dominant negative potency. Contrarily, equal amounts of c-erbA alpha 2 or hPPAR protein did not bind to either of the three response elements even in the presence of RXR. Since the TR:RXR heterodimers were only partially displaced from DNA in the presence of excess amounts of c-erbA alpha 2, it is likely that the TRE-unspecific dominant negative action of c-erbA alpha 2 is due in part to competition for DNA-binding and for TR-auxiliary proteins. In contrast, equimolar amounts of hPPAR completely inhibited the DNA-binding of hTR beta 1:RXR heterodimers, but not of TR:TR homodimers, suggesting that hPPAR has a higher RXR-binding affinity and is therefore a potent competitor for intranuclear RXR. Since thyroid hormones and peroxisome proliferators regulate in part a similar subset of target genes involved in fatty acid metabolism, these results suggest the possibility of cross-talk among the thyroid hormone and peroxisome proliferator signalling pathways. In summary, the results suggest that thyroid hormone action can be modulated by at least three different mechanisms: (i) increased binding of mutant hTRs to specific TREs; (ii) efficient competition for limiting amounts of RXR through the preferential formation of hPPAR:RXR, rather than TR:RXR heterodimers; and (iii) competition for binding to DNA and to auxiliary proteins other than RXR in the case of c-erbA alpha 2.
Mol Cell Endocrinol 1995 Jan
PMID:Modulation of thyroid hormone action by mutant thyroid hormone receptors, c-erbA alpha 2 and peroxisome proliferator-activated receptor: evidence for different mechanisms of inhibition. 779 35

Thyroid hormone (T3) stimulates Na,K-ATPase activity and alpha and beta subunit mRNA abundances in myocardial cells in vivo and in vitro. In this study, we used transient transfection and nuclear run-on assays to determine whether T3 regulates the transcription rate of the Na,K-ATPase alpha 2 subunit gene. Primary cultures of neonatal rat cardiac myocytes were incubated with 100 nM T3 for 1, 3, and 6 d, and alpha 2 mRNA levels were measured by Northern blot hybridization analysis. There was no change in the abundance of alpha 2 mRNA by 1 d of T3 treatment, whereas a two- and threefold increase in alpha 2 mRNA was evident when cells were exposed to T3 for 3 and 6 d, respectively. A portion of the rat alpha 2 gene containing 1700 base pairs (bp) of 5'-flanking DNA sequence was isolated and fused to the firefly luciferase gene. Transient transfection experiments utilizing this chimeric gene showed no T3 trans-activation of reporter gene activity either in the absence or presence of cotransfected beta 1 or alpha 1 isoforms of rat T3 receptor (T3R). In contrast, cotransfection of T3R facilitated a strong stimulation of luciferase activity driven by a construct containing a single copy of a palindromic T3 response element (TRE). Nuclear run-on analysis indicated that the rate of transcription of the endogenous alpha 2 gene was enhanced 1.2-fold at 3 d of T3 treatment, and was not regulated at either 1 or 6 d. These results indicate that the T3-dependent increase in alpha 2 mRNA content at 6 d is mediated at a post-transcriptional level. Unexpectedly, we observed a T3-dependent three-to sixfold repression of alpha 2/luciferase expression in cardiac myocytes cotransfected with T3R. Deletion analysis of the 5' end of the alpha 2 gene revealed a negative TRE between nucleotides -354 and -100.
Cell Mol Biol Res 1994
PMID:Thyroid hormone regulation of Na,K-ATPase alpha 2 gene expression in cardiac myocytes. 780 25

The paper reviews the current evidence on the role of thyroid hormones in regulating the creatine kinase energy transfer system at multiple structures in cardiac cells. 1) Thyroid hormones modulate the overall synthesis of phosphocreatine (PCr) by increasing the rate of mitochondrial oxidative phosphorylation. 2) Thyroid hormones regulate the total activity of creatine kinase and its isoenzyme distribution. In comparison with normal thyroid state (euthyroidism), hypothyroidism is characterized by decreased total creatine kinase activity owing to diminished fraction of creatine kinase. On the other hand, hyperthyroidism, while causing no change in total creatine kinase activity, leads to increased fractions of neonatal isoforms of creatine kinase, and, in case of prolonged hyperthyroidism, to decreased fraction of mitochondrial creatine kinase. The latter change is associated with partial uncoupling between mitochondrial creatine kinase and adenine nucleotide translocase reflected by decreased PCr/O ratio. 3) Hyperthyroidism leads to increased passive sarcolemmal permeability due to which the leakage of creatine along its concentration gradient occurs. As a result of (i) increased sarcolemmal permeability for creatine, (ii) uncoupling of mitochondrial PCr synthesis, and (iii) increased energy utilization rate the steady state intracellular PCr content decreases under hyperthyroidism which, in turn, increases the myocardial susceptibility to hypoxic damage. Thyroid state also modulates the protective effects of exogenous PCr on energetically depleted myocardium.
Mol Cell Biochem
PMID:Thyroid hormones and the creatine kinase system in cardiac cells. 780 61

The impact of type 1 diabetes mellitus on liver gamma-glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, gamma-glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13.2 fold in Sprague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, gamma-glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5'-nucleotidase was found to be similar: 9-14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing gamma-glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T3 (0.6 micrograms/Kg) once a day and T4 (6.0 micrograms/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20-40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in gamma-glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold over control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in gamma-glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma.
Mol Cell Biochem 1994 Oct 26
PMID:The impact of type I diabetes on rat liver gamma-glutamyltranspeptidase. 786 3

Thyroid hormone deficiency has dramatic effects on rat brain maturation. The expression of genes encoding neurotrophins and the trk family of neurotrophin receptors has been evaluated in several brain regions of normal and of neonatal or adult hypothyroid rats to analyze whether they are subject to thyroid hormone action. We found that hypothyroidism decreased trk mRNA levels in its major site of expression, the striatum, on postnatal days 5 (P5; 45%) and 15 (P15; 25%) and also in adults (35%). In contrast, no differences in trkB or trkC mRNAs levels were observed in any brain region at studied ages. According to previous reports, p75LNGFR mRNA was elevated in hypothyroid cerebellum as compared to age-matched controls on P5 and P15. We have also observed a distinct pattern for neurotrophin genes. The level of NGF mRNA was 20-50% lower in the cortex, hippocampus, and cerebellum of hypothyroid rats on neonatal hypothyroid rats on P15 and also after adult-onset hypothyroidism. Treatment of neonatally-induced hypothyroid rats with a single injection of triiodothyronine led to the recovery of hippocampal but not cortex NGF mRNA levels to that of control animals. On the contrary, no differences in the relatively high expression of the two mRNAs encoding BDNF were observed in any brain area. In contrast to a recent report, we did not find a reduction in brain NT-3 mRNA levels in hypothyroid animals. If any, the effect of thyroid deficiency in the hippocampus and cortex seems to be an early upregulation of NT-3 expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Brain Res Mol Brain Res 1994 Dec
PMID:Expression of neurotrophins and the trk family of neurotrophin receptors in normal and hypothyroid rat brain. 789 8

Thyroid hormone receptor (TR) forms homo- and heterodimers on various thyroid hormone response elements (TREs). We wished to clarify the relationship of homo- and heterodimer binding to TREs and their trans-activation. We investigated binding characteristics in gel mobility shift assays using synthetic direct repeat (DR) TREs having the consensus motifs separated by different oligonucleotide gaps, and we compared binding to trans-activation mediated via the direct repeat TRE. HTR alpha 1 purified from E. coli formed a monomer and homodimer on DR-TRE +0 to +5 but binding did not closely correlate with T3-dependent trans-activation. When RXR alpha expressed in COS 1 cell was added to purified TR alpha 1 in the gel shift assays, TR/RXR heterodimers were formed, and binding of heterodimers correlated highly with the level of trans-activation. These results strongly suggest that TR/TRAP heterodimers mediate the effect of thyroid hormone on DR-TREs. We also found T3-dependent disruption of homodimer formation on DR +0 to +2 and that T3 increased heterodimer formation on these TREs.
Mol Cell Endocrinol 1994 Jun
PMID:Differential binding and activation of thyroid hormone response elements by TR alpha 1 and RXR alpha-trap heterodimers. 792 63

Thyroid hormone (T3) receptor (TR) is a ligand-dependent transcription factor that acts through specific binding sites in the promoter region of target genes. In order to identify new genes that are regulated by T3, we used anti-TR antiserum to immunoprecipitate TR-DNA complexes from GH4 cell nuclei that had previously been treated with a restriction enzyme. Screening of the immunopurified, cloned DNA for TR binding sites by electrophoretic mobility shift assay yielded 53 positive clones. A subset of these clones was specifically immunoprecipitated with anti-TR antiserum and may therefore represent biologically significant binding sites. One of these clones, clone 122, was characterized in detail. It includes sequences highly related to the NICER long terminal repeat-like element and contains three TR binding sites as determined by DNase I footprinting. Two of the clone 122 TR binding sites are located upstream of the TATA box, and one is located downstream. The TR binding site downstream from the promoter was necessary and sufficient to confer T3-dependent regulation in transient transfection experiments. Expression of a reporter construct under the control of the clone 122 promoter region was activated by TR in the absence of ligand and returned to basal levels after T3 addition. Clone 122 sequences hybridize to at least two different mRNAs of approximately 6 and 10 kb from GH4 cells. The levels of both of these mRNAs increased upon removal of T3. Our studies suggest that specific immunoprecipitation of chromatin allows identification of binding sites and target genes for transcription factors.
Mol Cell Biol 1994 Nov
PMID:Isolation of a thyroid hormone-responsive gene by immunoprecipitation of thyroid hormone receptor-DNA complexes. 793 76

Thyroid hormone (T3) and retinoic acid (RA) are essential for normal vertebrate development and are known to coregulate several genes. Early development is predominantly retinoic acid sensitive, yet thyroid hormone receptor-alpha (T3R alpha) is expressed along with retinoic acid receptors (RAR)-alpha, -beta, and -gamma. To determine the role of unliganded T3R alpha in early development and on RA-stimulated neural development, we used homologous recombination techniques to inactivate both T3R alpha gene alleles in mouse embryonic stem (ES) cells. Loss of both T3R alpha alleles resulted in an increase in basal and RA-induced expression of the endogenous RA-responsive genes, RAR beta and alkaline phosphatase, which demonstrates that T3R alpha has an inhibitory effect on the RA response. A similar magnitude of T3R inhibition of the RA response was seen in transient transfection assays of RA response elements in both ES and assays of RA response elements in both ES and JEG cells. Cotransfection experiments were used to demonstrate that inhibition of the RA response could be mediated by T3R alpha 1. The addition of T3R alpha 1, but not the T3R alpha variant c-erbA alpha 2, to T3R alpha-null ES cells restored the inhibitory effect on RA-induced gene expression. RA-stimulated neural differentiation was seen in the wild-type, but not in T3R alpha-null ES, cells, consistent with reports of abnormal neural development as a consequence of premature RA stimulation. Our results demonstrate that the early expression of unliganded T3R alpha functions to modulate the RA response and RA-stimulated neural differentiation.
Mol Endocrinol 1994 Jun
PMID:Thyroid hormone receptor-alpha inhibits retinoic acid-responsive gene expression and modulates retinoic acid-stimulated neural differentiation in mouse embryonic stem cells. 793 90

Thyroid hormones and insulin regulate numerous cell processes and potentially interact through the transcriptional regulation of key genes. For instance, thyroid hormones stimulate the transcription of the fatty acid synthase and malic enzyme genes in chick embryonic hepatocytes, while insulin amplifies these effects. It is possible that insulin augments these actions of thyroid hormone by stimulating production of the thyroid hormone nuclear receptor (TR). In these studies, we examined the regulation of TR production/gene expression by insulin in bovine aortic endothelial cells (BAEC). We demonstrate that insulin significantly stimulates the gene expression of the TR alpha receptor, from BAEC. Insulin causes a maximal threefold induction above control TR alpha steady state mRNA levels in time and dose-related fashion in these cells. The increased mRNA mainly resulted from a twofold increase in transcription, as determined by nuclear run on. Insulin also increases thyroid receptor number and thyroid hormone binding, determined by Scatchard analysis of competitive inhibition binding studies. An established observation is that insulin can synergistically augment thyroid hormone-induced transcriptional activation of several important genes. It has also been previously determined that thyroid hormone action correlates closely to TR nuclear receptor number. Therefore, our studies, which show that insulin stimulates TR alpha production, suggests a potential mechanism whereby insulin can augment thyroid hormone transcriptional action.
Mol Cell Endocrinol 1994 Jul
PMID:Insulin stimulates thyroid hormone receptor alpha gene expression in cultured bovine aortic endothelial cells. 795 99


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