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W W Chin

Publications and source records attributed to W W Chin.

At least 91 records · Page 5Linked to original sources
PubMed1995

Studies on the repression of basal transcription (silencing) by artificial and natural human thyroid hormone receptor-beta mutants.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. Interestingly, in the absence of ligand, TRs also can repress basal transcription of positively regulated target genes, suggesting that unliganded TR may have a distinct role in gene regulation. In this paper, DNA binding, truncation, and natural human TR beta mutants were used in cotransfection and electrophoretic mobility shift assays to study various aspects of TR-mediated basal repression. Presently, little is known about the role(s) of natural human TR beta mutants on basal repression. These results show that: 1) TR binding to DNA likely is required for basal repression; 2) the amino-terminal region of TR is not required for basal repression; 3) TR homodimer binding is not absolutely required for basal repression, as TR mutants that selectively form TR-retinoid X receptor heterodimers can mediate basal repression; and 4) TR mutants with poor T3-binding affinity likely have constitutive basal repression, even in the presence of ligand. These findings provide new insight on the mechanism of basal repression by unliganded TRs.

Base Sequence↗
PubMed1995

Do clinical manifestations of resistance to thyroid hormone correlate with the functional alteration of the corresponding mutant thyroid hormone-beta receptors?

Resistance to thyroid hormone (RTH), a syndrome characterized by variable tissue hyposensitivity to thyroid hormone, is linked to mutations in the thyroid hormone receptor-beta (TR beta) gene. The purpose of this study was to determine whether the clinical phenotypes of RTH can be translated in terms of functional impairment of the corresponding mutant TR beta. Data from 124 subjects with RTH representing 18 different mutant TR beta s, showed that serum free T4 levels correlated with the degree of T3-binding impairment of the corresponding TR beta in 12 of these mutant TR beta s (group I), but not in the remaining 6 (group II). In subjects from both groups studied in detail by the administration of incremental doses of T3, the degree of thyrotroph resistance to T3 correlated with the magnitude of endogenous free T4 elevation at baseline, but did not parallel the resistance of peripheral tissues. In transfection studies, all group I mutant TR beta s inhibited positive transactivation by the wild type TR beta s to a similar degree in the presence of 1 nmol/L T3, whereas group II mutant TR beta s exerted a weaker inhibition that was not related to their T3-dependent trans-activation when tested alone. Similar results were obtained with negatively regulated reporter genes. It is concluded that the clinical severity of RTH, determined by thyrotroph resistance, can be predicted from the degree of T3 binding impairment and dominant negative potency of mutant TR beta s, but the degree of peripheral tissue resistance and related clinical manifestations is limited by putative genetic or environmental factors that modulate the effect of thyroid hormone.

Adolescent↗
PubMed1995

Regulated specific protein binding to a conserved region of the 3'-untranslated region of thyrotropin beta-subunit mRNA.

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.

Animals↗
PubMed1994

Hyperosmotic urea increases transcription and synthesis of Egr-1 in murine inner medullary collecting duct (mIMCD3) cells.

It was previously shown that when cells of renal epithelial origin are exposed to hyperosmotic urea in concentrations unique to the renal medulla, abundance of mRNA encoding the immediate-early gene product Egr-1 is up-regulated. This phenomenon appears restricted to cells of renal epithelial origin. In the present study, the newly isolated murine renal inner medullary cell line mIMCD3 was used to determine whether the urea-induced increase in Egr-1 mRNA abundance is associated with increased expression of functional protein product, and whether this increase is transcriptionally mediated. In Western analysis, urea (200 mM) increased Egr-1 immunoreactivity 3-fold relative to sham-treated cells. [35S]Methionine pulse-labeling followed by immunoprecipitation confirmed that this increased immunoreactivity was associated with increased de novo Egr-1 protein synthesis. Electrophoretic mobility shift assay demonstrated that urea treatment induced a commensurate increase in specific DNA binding activity for the Egr-1 consensus sequence. In addition, the increase in Egr-1 mRNA expression accompanying urea treatment was a consequence of enhanced transcription, as determined by nuclear run-off assay. Taken together, these data indicate that hyperosmotic urea increases both Egr-1 transcription and new protein synthesis in renal epithelial cells in culture, and that this newly synthesized Egr-1 is a functional DNA-binding protein. To our knowledge, this is the first example of urea-inducible gene transcription. In addition, Egr-1 represents the first eukaryotic transcription factor transcriptionally activated by a hyperosmotic stressor.

Animals↗
PubMed1994

Half-site arrangement of hybrid glucocorticoid and thyroid hormone response elements specifies thyroid hormone receptor complex binding to DNA and transcriptional activity.

Thyroid hormone receptors bind to thyroid hormone response elements (TREs) as heterodimers with 3,5,3'-L-triiodothyronine (T3) receptor auxiliary protein (TRAP) and retinoid X receptors (RXRs). Currently, it is not known whether TR/TRAP or TR/RXR heterodimers need to bind to both TRE half-sites and whether there is a preferred orientation for TR/RXR heterodimer binding to TREs or transcriptional activation. Accordingly, we created a mutant TR alpha (TR-P box) by changing 3 amino acids in the P box region of the first zinc finger of the DNA-binding domain to that of the glucocorticoid receptor (GR), and we examined wild-type TR alpha and TR-P box complex binding to hybrid response elements containing TRE and glucocorticoid receptor element (GRE) half-sites arranged as a direct repeat with a four-nucleotide gap. TR-P box/RXR heterodimers selectively bound to the hybrid response elements in which GRE half-site was the downstream half-site, whereas TR alpha/RXR bound to hybrid response elements in which GREs were in either position. Additionally, TR/TRAP or TR/RXR heterodimer required two half-sites for binding to DNA, with strong binding to at least one of the half-sites. Last, co-transfection assays and methylation interference studies using the hybrid response elements suggest that the sequential arrangement of strong and weak half-sites in the TRE may be a critical determinant of TR/RXR heterodimer binding and transcriptional activation.

Base Sequence↗
PubMed1994

Roles of v-erbA homodimers and heterodimers in mediating dominant negative activity by v-erbA.

v-erbA, a viral oncogenic homolog of thyroid hormone receptor (TR), blocks the effect of T3 in TR-mediated transcription. The mechanism(s) for this dominant negative effect by v-erbA on TRs is unknown but may involve competition between v-erbA and TR-containing complexes for binding to thyroid hormone response elements (TREs) and/or protein-protein interactions between v-erbA and TR. To investigate these potential mechanisms, we used the electrophoretic mobility shift assay to compare in vitro translated v-erbA and TR alpha binding to two TREs-chick lysozyme TRE (F2) and direct repeat TRE (DR4). v-erbA bound as a homodimer to these TREs, whereas TR alpha bound as a homodimer and monomer. T3 decreased TR alpha homodimer binding to the TREs as we reported previously; however, surprisingly, high concentrations of T3 (10(-6) M) also decreased v-erbA homodimer binding to the TREs. Additionally, v-erbA formed heterodimers with nuclear proteins such as retinoid X receptor and T3 receptor auxiliary protein as well as with TR alpha. These dimers remained bound to DNA in the presence of T3. Finally, v-erbA could not mediate ligand-dependent transcriptional activation even at 10(-6) M T3 but could block ligand-dependent TR-mediated transactivation in co-transfection experiments. v-erbA also exhibited differential dominant negative activity on F2 and DR4 suggesting that half-site sequence and/or orientation may influence v-erbA-dominant negative activity. In sum, there are multiple v-erbA complexes that bind to TREs in the presence of T3, which all may contribute to v-erbA's dominant negative effect on TR-mediated transcription by competing with TR-containing complexes for binding to TREs.

Base Sequence↗
PubMed1994

In vitro transcriptional studies of the roles of the thyroid hormone (T3) response elements and minimal promoters in T3-stimulated gene transcription.

Thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that are encoded by two different genes, TR alpha and TR beta, and bind to thyroid hormone response elements (TREs) in the promoters of thyroid hormone (T3)-regulated genes. Retinoid X receptors (RXRs), major members of the thyroid hormone receptor auxiliary proteins, have recently been shown to enhance the binding of TRs to TREs. We previously showed that TRs extracted from rat pituitary GH3 cells retain ligand (T3) and DNA binding specificity and stimulate rat growth hormone (rGH) promoter activity in a cell-free in vitro transcription system. In this report, we have studied further how T3 activates endogenous TRs and stimulates transcription from different TRE-containing promoters. We found that T3 (10(-8) M) selectively stimulates transcription from rGH-TRE- and TREpal-, but not ME-TRE- and F2-TRE-, containing templates in which these TREs are linked in front of the rGH minimal promoter containing only the TATA box binding protein, but not any other proximal binding protein, sequence. In contrast, only the TREpal/AdML template, in which TREpal oligonucleotide was linked in front of the adenovirus major late gene (AdML) minimal promoter, was stimulated by T3. Electrophoretic mobility shift assay (EMSA) demonstrates that endogenous TR complexes specifically bind to either natural or idealized TRE (rGH-TRE, TREpal, ME-TRE, and F2-TRE) oligonucleotides. To further understand these receptor-DNA complexes formed on various TREs, isoform-specific anti-receptor antisera (TR alpha, TR beta 1, TR beta 2, and RXR beta) were added in the EMSA. These antisera differentially supershifted TR.DNA complexes formed on the TREs. These data suggest either that endogenous TR isoforms and RXR beta may form different complexes on the various TREs or that TR.RXR complexes have distinct conformations when bound to the various TREs. Taken together, these data suggest that particular TREs in which specific TR.RXR complexes are formed and different minimal promoters may provide specificity in T3-mediated transcriptional stimulation of gene expression.

Animals↗
PubMed1994

Phosphorylation selectively increases triiodothyronine receptor homodimer binding to DNA.

Thyroid hormone receptors (TRs) are ligand-regulated transcription factors that bind to thyroid hormone response elements (TREs) as monomers and homodimers, and as heterodimers with nuclear proteins such as TR auxiliary proteins and retinoid X receptors. Recently, bacterially expressed human TR beta-1 (hTR beta-1) was shown to be phosphorylated in vitro by HeLa cytosolic extract. However, little is known about the consequences of phosphorylation on the nature of TR complexes. Therefore, we studied the effect of phosphorylation on TR binding of TREs. Bacterially expressed hTR beta-1 was phosphorylated in vitro with ATP by HeLa cytosolic extract. The ratio of phosphoserine to phosphothreonine was approximately 5:1. We then analyzed phosphorylated hTR beta-1 binding to several TREs by electrophoretic mobility shift assay. Phosphorylated hTR beta-1 bound better as a homodimer to the TREs than hTR beta-1 incubated with preheated cytosolic extract. Alkaline phosphatase treatment of the phosphorylated hTR beta-1 eliminated the enhanced homodimer binding to DNA. In contrast, phosphorylation did not affect TR/TR auxiliary protein or TR/retinoid X receptor heterodimer binding to DNA. Triiodothyronine decreased both phosphorylated and unphosphorylated hTR beta-1 homodimer binding to several TREs, and the addition of okadaic acid did not alter this triiodothyronine effect. These results indicate that phosphorylation, in addition to ligand binding, modulates TR dimer binding to TREs. As such, it is possible that phosphorylation may also participate in TR-mediated regulation of transcription.

Blotting, Western↗
PubMed1994

Chromosomal localization of the gonadotropin-releasing hormone receptor gene to human chromosome 4q13.1-q21.1 and mouse chromosome 5.

The gonadotropin-releasing hormone receptor (GRHR) is a G-protein-coupled receptor on the cell surface of pituitary gonadotropes, where it serves to transduce signals from the extracellular ligand, the hypothalamic factor gonadotropin-releasing hormone, and to modulate the synthesis and secretion of luteinizing hormone and follicle-stimulating hormone. We have localized the GRHR gene to the q13.1-q21.1 region of the human chromosome 4 using mapping panels of human/rodent somatic cell hybrids containing different human chromosomes or different regions of human chromosome 4. Furthermore, using linkage analysis of single-strand conformational polymorphisms, the murine GRHR gene was localized to mouse chromosome 5, linked to the endogenous retroviral marker Pmv-11. This is consistent with te evolutionary conservation of homology between these two regions, as has been previously suggested from comparative mapping of several other loci. The localization of the GRHR gene may be useful in the study of disorders of reproduction.

Animals↗
PubMed1994

Molecular mechanisms of thyroid hormone action.

This brief review deals with the key trans- and cis-elements that are currently believed to participate in TH action at the transcriptional level. The reader is also referred to other recent reviews (53,54). In sum, there are multiple levels of complexity that make an understanding of the fundamental mechanisms of TH action difficult. However, the interactions of multiple TRs and TRAPS along with putative coactivators and ligands provide the cell with a wide range of biological responses. An appreciation of these phenomena will ensure insight into regulated gene expression in general, and the mode of action of TH in particular. While the field has progressed greatly since Jack Oppenheimer first described the nuclear binding of TH, there are "many more miles to go" for all of us, including Jack. We trust the journey will continue to be captivating.

Animals↗
PubMed1994

Evidence that phosphorylation events participate in thyroid hormone action.

Using both a protein phosphatase inhibitor, okadaic acid (OA), and a protein kinase inhibitor, H7, to modify phosphorylation events in the cell, we investigated the effects of these agents on transcriptional activation via exogenous rat thyroid hormone receptor (TR) isoforms in transiently transfected cells and endogenous TRs. CV-1 cells were transiently cotransfected with expression plasmids encoding either the rat TR alpha 1 or TR beta 1, and luciferase reporter plasmids containing either the synthetic DR4 or the chick lysozyme F2 thyroid hormone response elements (TREs). For both receptor isoforms, there was an enhancement of transcriptional activity after incubation with 5 nM T3 for 24 h compared to hypothyroid levels. There was little change in transcriptional activation in the presence of 25 nM OA alone; however, for both TR isoforms and both TREs studied, OA augmented the stimulatory effects of T3. For the F2 TRE, transcriptional activation via TR alpha 1 increased from 19- to 35-fold, and that via TR beta 1 increased from 6- to 10-fold in the presence of T3 and OA compared to that with T3 alone. Similar results were found for the DR4 TRE. OA enhanced transcriptional activation by T3 in a dose-dependent manner. Increasing concentrations of OA (0, 5, 25, and 50 nM) further increased relative luciferase activity from 11-fold in the absence of OA to 45-fold in the presence of 50 nM OA. The protein kinase inhibitor, H7, caused no change in the transcriptional activity of the reporter plasmids via TR alpha 1 in the absence of T3, but completely blocked transcriptional activation by T3 for both the DR4 and the F2 TREs. H7 also blocked stimulation of endogenous GH and inhibition of endogenous TR beta 2 mRNAs by T3 in GH3 cells. These results indicate that phosphorylation events in the cell play an important role in transcriptional activation via both TR isoforms.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Roles of 3,5,3'-triiodothyronine and deoxyribonucleic acid binding on thyroid hormone receptor complex formation.

Thyroid hormone receptors (TRs) bind to thyroid hormone response elements (TREs) in the promoter region of target genes as monomers, homodimers, and heterodimers with nuclear proteins such as retinoid-X receptors (RXRs). Recently, we observed that T3 decreased TR homodimer, but not TR/RXR heterodimer, binding to TREs, suggesting that the latter complexes may be involved in transcriptional activation of target genes. However, little is known about TR complexes that form in solution. Thus far, there have been only limited studies comparing TR complex formation in solution and on DNA as well as examining the effects of T3 and the putative ligand for RXRs, 9-cis retinoic acid (9-cis RA), on TR complex formation. In this paper, we used a coimmunoprecipitation assay with anti-TR beta 1 antibody and the electrophoretic mobility shift assay under similar buffer and incubation conditions to demonstrate that in the absence of T3, TR beta 1 is present as a monomer in solution and binds primarily as a homodimer to the chicken lysozyme TRE, F2. In the presence of T3, TR beta 1 cannot form a homodimer on F2, but, instead, exists as a liganded monomer in solution. Kinetic studies demonstrated that T3 markedly increased the dissociation rate of TR homodimer from F2. Using similar methods, we observed TR beta 1/RXR alpha heterodimer formation in solution and 10-fold greater formation on F2. Neither T3 nor 9-cis RA significantly affected TR beta 1/RXR alpha heterodimer formation. Taken together, these results suggest that both T3 and TRE binding are important determinants of the formation of specific TR complexes in solution and on DNA.

Base Sequence↗
PubMed1994

Stable transfection of GH3 cells with rat gonadotropin-releasing hormone receptor complementary deoxyribonucleic acid results in expression of a receptor coupled to cyclic adenosine 3',5'-monophosphate-dependent prolactin release via a G-protein.

GH3 cells, which normally release PRL in response to stimulation by TRH, have been stably transfected with rat GnRH receptor complementary DNA (GGH3-1' cells). Unlike the parent line, GGH3-1' cells express GnRH receptor, which can be measured in a radioligand assay using a metabolically stable GnRH analog. The number of receptors (11,000 +/- 2,800 receptors/cell; n = 3) and Kd (4.1 +/- 1.0 x 10(-8) M; n = 3), determined using a radioiodinated GnRH agonist, as well as binding inhibition values for GnRH agonists and antagonists and for unrelated substances suggest that this receptor is similar to those expressed in cell cultures derived from rat pituitaries, although the binding affinity is about 1 log lower in the former. Unlike GnRH-stimulated release of gonadotropins from primary pituitary cultures, which does not require protein synthesis and is not coupled to cAMP production, GnRH-stimulated PRL release from the transfected cell line is absolutely dependent on protein synthesis, and cAMP fulfills the requirements of a second messenger. The receptor appears to be coupled to adenylate cyclase-mediated PRL release through a cholera toxin-sensitive G-protein. These studies provide functional evidence to support the view that the cloned receptor is the physiological receptor for the releasing hormone, and that this receptor can differentially couple to G-proteins depending on their availability and accessibility in the target cell.

Animals↗
PubMed1994

Differential expression of thyroid hormone receptor isoforms in neurons and astroglial cells.

The brain has abundant nuclear T3-binding sites and contains messenger RNAs (mRNAs) encoding multiple thyroid hormone receptor (TR) isoforms; the cellular distribution of these different TR isoforms is unknown. To determine whether the TR isoforms are differentially expressed in neuronal and astroglial cells, we examined the relative abundance of the mRNAs encoding TR alpha 1, c-erbA alpha 2, and TR beta 1 in primary cultures of fetal rat brain and in several cell lines of neural and glial origin. Additionally, the TR isoform polypeptides were identified by immunocytochemistry using isoform-specific antibodies. Northern blot analysis showed that fetal brain cell cultures contain mRNAs encoding the T3-binding isoforms TR alpha 1 and TR beta 1 as well as the mRNA encoding the non-T3-binding c-erbA alpha 2. c-erbA alpha 2 mRNA was most abundant, comprising more than 85% of the TR mRNAs in the primary cultures. Neuronal enrichment by antimitotic selection increased TR beta 1 mRNA approximately 3-fold, decreased c-erbA alpha 2 mRNA 70%, and had little or no effect on TR alpha 1 mRNA. Neuronal depletion resulted in the complete loss of TR beta 1 mRNA without changing c-erb alpha 2 or TR alpha 1 mRNA levels. Primary cultures of rat astrocytes, the astrocytoma cell line C6, and the pheochromocytoma cell line PC12 contained only the c-erbA alpha 2 mRNA. Immunocytochemistry using isoform-specific anti-sera revealed that TR beta 1 was exclusively localized to neuronal nuclei, and c-erbA alpha 2 was only found in the nuclei of astrocytes. These results show that TR beta 1 is localized to the nuclei of neuronal cells, and that c-erbA alpha 2 is restricted to the nuclei of astrocytes.

Animals↗
PubMed1994

Thyroid hormone receptor monomer, homodimer, and heterodimer (with retinoid-X receptor) contact different nucleotide sequences in thyroid hormone response elements.

Thyroid hormone receptors (TRs) bind as monomers, homodimers, and heterodimers with nuclear proteins such as retinoid-X receptors (RXRs) to thyroid hormone response elements (TREs). However, it is not known which nucleotides each TR complex contacts in a particular TRE. To identify the precise contact sites on a synthetic DR4 (TRE half-sites arranged as a direct repeat with a four-nucleotide spacer) and a chick lysoenzyme TRE, F2 (half-sites arranged as an inverted palindrome with a six-nucleotide spacer), for various TR complexes, mobility shift assays, and dimethylsulfate and KMnO4 DNA modification interference assays were employed. First, TR alpha monomer bound to the downstream half-site of these TREs, whereas TR alpha homodimer bound to both half-sites. TR alpha/RXR alpha heterodimer also bound to both half-sites, but "preferred" to contact the down-stream half-site. Second, the specific flanking and spacing sequences of DR4 influenced the contact sites and the binding of TR alpha monomer and homodimer, but not TR alpha/RXR alpha heterodimer. Finally, cotransfection studies, using reporter plasmids containing DR4 or F2 in both orientations with respect to the basal promoter, provide evidence that preferential contact with the down-stream half-site by TR/RXR heterodimer may be important for maximal transcriptional activation.

Animals↗