The type IIa renal sodium-dependent phosphate (Na/P i ) co-transporter Npt2a is implicated in the control of serum phosphate levels. It has been demonstrated previously that renal Npt2a protein and its mRNA expression are both up-regulated by the thyroid hormone T 3 (3,3′,5-tri-iodothyronine) in rats. However, it has never been established whether the induction was mediated by a direct effect of thyroid hormones on the Npt2a promoter. To address the role of Npt2a in T 3 -dependent regulation of phosphate homoeostasis and to identify the molecular mechanisms by which thyroid hormones modulate Npt2a gene expression, mice were rendered pharmacologically hypo- and hyper-thyroid. Hypothyroid mice showed low levels of serum phosphate and a marked decrease in renal Npt2a protein abundance. Importantly, we also showed that Npt2a-deficient mice had impaired serum phosphate responsiveness to T 3 compared with wild-type mice. Promoter analysis with a luciferase assay revealed that the transcriptional activity of a reporter gene containing the Npt2a promoter and intron 1 was dependent upon TRs (thyroid hormone receptors) and specifically increased by T 3 in renal cells. Deletion analysis and EMSAs (electrophoretic mobility-shift assays) determined that there were unique TREs (thyroid-hormone-responsive elements) within intron 1 of the Npt2a gene. These results suggest that Npt2a plays a critical role as a T 3 -target gene, to control phosphate homoeostasis, and that T 3 transcriptionally activates the Npt2a gene via TRs in a renal cell-specific manner.

Selenoproteins are proteins containing selenium in the form of the 21st amino acid, selenocysteine. Members of this protein family have many diverse functions, but their synthesis is dependent on a common set of cofactors and on dietary selenium. Although the functions of many selenoproteins are unknown, several disorders involving changes in selenoprotein structure, activity or expression have been reported. Selenium deficiency and mutations or polymorphisms in selenoprotein genes and synthesis cofactors are implicated in a variety of diseases, including muscle and cardiovascular disorders, immune dysfunction, cancer, neurological disorders and endocrine function. Members of this unusual family of proteins have roles in a variety of cell processes and diseases.

RNA possesses the ability to bind a wide repertoire of small molecules. Some of these binding interactions have been shown to be of primary importance in molecular biology. For example, several classes of mRNA domains, collectively referred to as riboswitches, have been shown to serve as RNA genetic control elements that sense the concentrations of specific metabolites (i.e. acting as direct sensors of chemical compounds). However, to date no RNA species binding a hormone has been reported. Here, we report that the use of an appropriate SELEX (systematic evolution of ligands by exponential enrichment) strategy results in the isolation of thyroxine-specific aptamers. Further biochemical characterization of these aptamers, including mutational studies, the use of transcripts with site-specific modified nucleotides, nuclease and chemical probing, binding-shift assays and CD, demonstrated that these RNA structures included a G-rich motif, reminiscent of a guanine quadruplex structure, adjacent to a helical region. The presence of the thyroxine appeared to be essential for the formation of the structural motif's scaffold. Moreover, the binding is shown to be specific to thyroxine (T4) and tri-iodothyronine (T3), the active forms of the hormone, whereas other inactive derivatives, including thyronine (T0), do not support complex formation. These results suggest that this aptamer specifically binds to the iodine moieties of the thyroxine, a previously unreported ability for an RNA molecule.

The transcription of the human UCP3 (uncoupling protein-3) gene in skeletal muscle is tightly regulated by metabolic signals related to fatty acid availability. However, changes in thyroid status also modulate UCP3 gene expression, albeit by unknown mechanisms. We created transgenic mice bearing the entire human UCP3 gene to investigate the effect of thyroid hormones on human UCP3 gene expression. Treatment of human UCP3 transgenic mice with thyroid hormones induced the expression of the human gene in skeletal muscle. In addition, transient transfection experiments demonstrate that thyroid hormones activate the transcription of the human UCP3 gene promoter when MyoD and the TR (thyroid hormone receptor) were co-transfected. The action of thyroid hormones on UCP3 gene transcription is mediated by the binding of the TR to a proximal region in the UCP3 gene promoter that contains a direct repeat structure. An intact DNA sequence of this site is required for thyroid hormone responsiveness and TR binding. Chromatin immunoprecipitation assays revealed that the TR binds this element in vivo . The murine Ucp3 gene promoter was also dependent on MyoD and responsive to thyroid hormone in transient transfection assays. However, it was much less sensitive to thyroid hormone than the human UCP3 promoter. In summary, UCP3 gene transcription is activated by thyroid hormone treatment in vivo , and this activation is mediated by a TRE (thyroid hormone response element) in the proximal promoter region. Such regulation suggests a link between UCP3 gene expression and the effects of thyroid hormone on mitochondrial function in skeletal muscle.

The molecular mechanism involved in the liganded thyroid hormone receptor suppression of the TSHβ (thyroid-stimulating hormone β, or thyrotropin β) gene transcription is undetermined. One of the main reasons is the limitation of useful cell lines for the experiments. We have developed an assay system using non-pituitary CV1 cells and studied the negative regulation of the TSHβ gene. In CV1 cells, the TSHβ–CAT (chloramphenicol acetyltransferase) reporter was stimulated by Pit1 and GATA2 and suppressed by T 3 (3,3´,5-tri-iodothyronine)-bound thyroid hormone receptor. The suppression was dependent on the amounts of T 3 and the receptor. Unliganded receptor did not stimulate TSHβ activity, suggesting that the receptor itself is not an activator. Analyses using various receptor mutants revealed that the intact DNA-binding domain is crucial to the TSHβ gene suppression. Co-activators and co-repressors are not necessarily essential, but are required for the full suppression of the TSHβ gene. Among the three receptor isoforms, β2 exhibited the strongest inhibition and its protein level was the most predominant in a thyrotroph cell line, TαT1, in Western blotting. The dominant-negative effects of various receptor mutants measured on the TSHβ–CAT reporter were not simple mirror images of those in the positive regulation under physiological T 3 concentration.

The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D 3 . Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Björkhem (1997) J. Biol. Chem. 272 , 26253–26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5′-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2–3-fold, whereas thyroxine (T 4 ) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T 4 and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position −1094 bp. The putative response elements for dexamethasone were mapped to positions between −792 and −1095 bp. The −451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T 4 and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.

We identified a thyroid hormone [3,5,3′-tri-iodothyronine (T 3 )]-responsive gene, ZAKI-4, in cultured human skin fibroblasts. It belongs to a family of genes that encode proteins containing a conserved motif. The motif binds to calcineurin and inhibits its phosphatase activity. In the present study, we have demonstrated three different ZAKI-4 transcripts, α, β1 and β2, in human brain by 5′- and 3′-RACE (rapid amplification of cDNA ends). The α transcript was identical with the one that we originally cloned from human fibroblasts and the other two are novel. The three transcripts are generated by alternative initiation and splicing from a single gene on the short arm of chromosome 6. It is predicted that β1 and β2 encode an identical protein product, β, which differs from α in its N-terminus. Since α and β contain an identical C-terminal region harbouring the conserved motif, both isoforms are suggested to inhibit calcineurin activity. Indeed, each isoform associates with calcineurin A and inhibits its activity in a similar manner, suggesting that the difference in N-terminus of each isoform does not affect the inhibitory function on calcineurin. An examination of the expression profile of the three transcripts in 12 human tissues revealed that the α transcript is expressed exclusively in the brain, whereas β transcripts are expressed ubiquitously, most abundantly in brain, heart, skeletal muscle and kidney. It was also demonstrated that human skin fibroblasts express both α and β transcripts, raising the question of which transcript is up-regulated by T 3 . It was revealed that T 3 markedly induced the expression of α isoform but not of β. This T 3 -mediated increase in the α isoform was associated with a significant decrease in endogenous calcineurin activity. These results suggest that the expression of ZAKI-4 isoforms is subjected to distinct hormonal as well as tissue-specific regulation, constituting a complex signalling network through inhibition of calcineurin.

Treatment of rats with thyroxine has been shown to elevate the biosynthesis and content of cardiolipin in the heart [Cao, Cheng, Angel and Hatch (1995) Biochim. Biophys. Acta 1256, 241-244]. Treatment with thyroxine resulted in a 1.8-fold increase ( P < 0.025) in [1- 14 C]linoleate and a 1.7-fold increase ( P < 0.025) in [1- 14 C]oleate incorporated into cardiolipin in perfused hearts, compared with controls. The mechanism for the elevation in incorporation of unsaturated fatty acids into cardiolipin was a 1.6-fold ( P < 0.025) increase in mitochondrial monolysocardiolipin acyltransferase activity. The results demonstrate that the acylation of cardiac monolysocardiolipin is regulated by thyroid hormone. Thus an elevation in cardiolipin biosynthesis is accompanied by an elevation in monolysocardiolipin acyltransferase activity to maintain the appropriate molecular species composition of cardiolipin in the cardiac mitochondrial membrane. We postulate that monolysocardiolipin acyltransferase might be a rate-limiting enzyme for the molecular remodelling of cardiolipin in the heart.