Nuclear thyroid hormone receptors in cultured human fibroblasts: Improved method of isolation, partial characterization, and interaction with chromatin

Ichikawa, Kazuo; DeGroot, Leslie J.; Refetoff, Samuel; Horwitz, Allen L.; Pollak, Elizabeth R.

doi:10.1016/0026-0495(86)90229-5

Cited by 13 publications

(5 citation statements)

References 38 publications

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“…Phasecontrast microscopy revealed that the nuclear preparation was free of intact cells and had little cytoplasmic contamination and that integrity of the nuclei was well preserved; however, nuclei isolated in this way exhibited a separation of the outer nuclear membrane from the inner membrane on electron microscopy (Ichikawa et al 1986). To determine the binding of SK&F L-94901 to the nuclear receptor, isolated nuclei were incubated with 14·4 pM …”

Section: Binding Of Thyronine Analogs To Isolated Nucleimentioning

confidence: 99%

Mechanism of liver-selective thyromimetic activity of SK&F L-94901: evidence for the presence of a cell-type-specific nuclear iodothyronine transport process

Ichikawa¹,

Miyamoto²,

Kakizawa³

et al. 2000

Journal of Endocrinology

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The thyromimetic compound SK&F L-94901 shows more potent thyromimetic activity in the liver than in the pituitary gland or heart when administered to rats. The mechanisms of liver-selectivity of SK&F L-94901 were examined using cultured rat hepatoma cells (dRLH-84) and rat pituitary tumor cells (GH3), both of which showed saturable cellular uptake of tri-iodothyronine (T 3 ). When isolated nuclei with partial disruption of the outer nuclear membrane were used, SK&F L-94901 competed for [ 125 I]T 3 binding to nuclear receptors almost equally in dRLH-84 and GH3 cells. SK&F L-94901 also did not discriminate thyroid hormone receptors (TR) 1 and 1 in terms of binding affinity and activation of the thyroid hormone responsive element. In intact cells, however, SK&F L-94901 was a more potent inhibitor of nuclear [ 125 I]T 3 binding in dRLH-84 cells than in GH3 cells at an early phase of the nuclear uptake process and after binding equilibrium. These data suggest that SK&F L-94901 is more effectively transported to nuclear TRs in hepatic cells than in pituitary cells and therefore shows liverselective thyromimetic activity. In conclusion, SK&F L-94901 discriminates hepatic cells and pituitary cells at the nuclear transport process. The cellular transporters responsible for this discrimination were not evident.

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Section: Binding Of Thyronine Analogs To Isolated Nucleimentioning

confidence: 99%

Mechanism of liver-selective thyromimetic activity of SK&F L-94901: evidence for the presence of a cell-type-specific nuclear iodothyronine transport process

Ichikawa¹,

Miyamoto²,

Kakizawa³

et al. 2000

Journal of Endocrinology

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“…Identification of T 3 -responsive gene(s) from the tissues that maintain differentiated function is thus preferable. Human skin fibroblasts fulfill this requirement since they express T 3 -receptors (3)(4)(5)(6) and are responsive to T 3 . In cultured skin fibroblasts, we have shown that T 3 inhibits the synthesis of glycosaminoglycan (7,8) and fibronectin (9), and Chait et al (10) demonstrated that it enhances low density lipoprotein degradation.…”

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confidence: 99%

Molecular Cloning of a Novel Thyroid Hormone-responsive Gene, , in Human Skin Fibroblasts

Miyazaki

Kanou

Murata

et al. 1996

Journal of Biological Chemistry

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Utilizing a method called "differential display of mRNAs by means of polymerase chain reaction", the cDNA fragment of a thyroid hormone-responsive gene ZAKI-4 was cloned from cultured human skin fibroblasts. Northern blot analysis revealed that there were two ZAKI-4 mRNA species (3.4 and 1.4 kilobases (kb)), and they were up-regulated by a physiological concentration of triiodothyronine (T3). This T3 effect was abolished by the treatment with cycloheximide, indicating the possibility that gene ZAKI-4 is regulated by T3 in an indirect fashion, through an intermediate product of T3, rather directly by T3 itself. No effect of T3 on ZAKI-4 mRNA stability suggested that T3 induces the mRNA at the transcriptional level. Rapid amplification of cDNA ends confirmed the presence of two mRNA species. ZAKI-4 mRNA was detected in heart, brain, liver, and skeletal muscle but not in placenta, lung, kidney and pancreas. In skin fibroblasts and skeletal muscle, 3.4-kb mRNA was the major species, whereas 1.4-kb mRNA was dominant in heart, brain, and liver. The sequence analysis suggested that the two mRNA species arise from alternative polyadenylation and code a single protein of 192 amino acids. No homologous protein sequence was found in a data base. Elucidation of the function of ZAKI-4 gene product will provide new insights into an important role of T3 in various organs.

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“…The thyroid gland also produces smaller amounts of triiodothyronine (T 3 ; 3,5,3Ј-triiodo-L-thyronine) and reverse T 3 (rT 3 ; 3,3Ј,5Ј-triiodo-L-thyronine), and 80% of T 4 is converted to T 3 and rT 3 in peripheral tissues by two selenium deiodinases, which are tissue-specific (4). Current beliefs are that T 3 is the dominant active form of TH; T 3 binds the TRs with an affinity about 20 -30 times higher than that of T 4 (5)(6)(7)(8)(9), and some studies suggest that T 3 is present at higher concentrations in the nucleus than T 4 (10,11). Nonetheless, the question of whether T 4 is simply a prohormone or an active TH species is not completely resolved.…”

mentioning

confidence: 99%

Thyroxine-Thyroid Hormone Receptor Interactions

Sandler

Webb

Apriletti

et al. 2004

Journal of Biological Chemistry

122

118

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Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs ␣ and ␤) that bind triiodothyronine (T 3 , 3,5,3-triiodo-L-thyronine) with high affinity, and its precursor thyroxine (T 4 , 3,5,3,5-tetraiodo-L-thyronine) with lower affinity. T 4 contains a bulky 5 iodine group absent from T 3 . Because T 3 is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5 substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T 4 affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T 4 complexes adopt a conformation that differs from TR-T 3 complexes in solution. Nonetheless, T 4 behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T 3 does not contribute to agonist activity. We determined x-ray crystal structures of the TR␤ LBD in complex with T 3 and T 4 at 2.5-Å and 3.1-Å resolution. Comparison of the structures reveals that TR␤ accommodates T 4 through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5 iodine and complete the coactivator binding surface. While T 3 is the major active TH, our results suggest that T 4 could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5 extension should be considered in TR ligand design. Thyroid hormone (TH)1 plays important regulatory roles in metabolism, homeostasis, and development by binding and altering the transcriptional regulatory properties of two related nuclear receptors (NRs), the thyroid hormone receptors (TRs) ␣ and ␤ (1, 2). Most TH produced in the thyroid gland is secreted in the form of thyroxine (T 4 ; 3,5,3Ј,5Ј-tetraiodo-L-thyronine) (2, 3). The thyroid gland also produces smaller amounts of triiodothyronine (T 3 ; 3,5,3Ј-triiodo-L-thyronine) and reverse T 3 (rT 3 ; 3,3Ј,5Ј-triiodo-L-thyronine), and 80% of T 4 is converted to T 3 and rT 3 in peripheral tissues by two selenium deiodinases, which are tissue-specific (4). Current beliefs are that T 3 is the dominant active form of TH; T 3 binds the TRs with an affinity about 20 -30 times higher than that of T 4 (5-9), and some studies suggest that T 3 is present at higher concentrations in the nucleus than T 4 (10, 11). Nonetheless, the question of whether T 4 is simply a prohormone or an active TH species is not completely resolved. T 4 exerts rapid nongenomic effects at several loci distinct from TRs (12). Moreover, saturating levels of T 4 activate transcription of TH-responsive genes in cell culture (see for example Ref. 5). Whereas it is possible that at least some of this activity is due to T 3 generated from T 4 in the cell, these results suggest that T 4 may act as a TR agonist. Normal concentrations of plasma-free T 4 are about 4 -6-fold higher than th...

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Nuclear thyroid hormone receptors in cultured human fibroblasts: Improved method of isolation, partial characterization, and interaction with chromatin

Cited by 13 publications

References 38 publications

Mechanism of liver-selective thyromimetic activity of SK&F L-94901: evidence for the presence of a cell-type-specific nuclear iodothyronine transport process

Mechanism of liver-selective thyromimetic activity of SK&F L-94901: evidence for the presence of a cell-type-specific nuclear iodothyronine transport process

Molecular Cloning of a Novel Thyroid Hormone-responsive Gene, , in Human Skin Fibroblasts

Thyroxine-Thyroid Hormone Receptor Interactions

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