Profiling retrospective thyroid function data in complete thyroidectomy patients to investigate the HPT axis set point (PREDICT-IT).

BACKGROUND: The homeostatic euthyroid set point of the hypothalamus-pituitary-thyroid axis of any given individual is unique and oscillates narrowly within substantially broader normal population ranges of circulating free thyroxine (FT4) and thyroid-stimulating hormone (TSH), otherwise termed 'thyroid function test (TFT)'. We developed a mathematical algorithm codenamed Thyroid-SPOT that effectively reconstructs the personalized set point in open-loop situations and evaluated its performance in a retrospective patient sample. METHODS: We computed the set points of 101 patients who underwent total thyroidectomy for non-functioning thyroid disease using Thyroid-SPOT on each patient's own serial post-thyroidectomy TFT. Every predicted set point was compared against its respective healthy pre-operative euthyroid TFT per individual and their separation (i.e. predicted-observed TFT) quantified. RESULTS: Bland-Altman analysis to measure the agreement between each pair of an individual's predicted and actual set points revealed a mean difference in FT4 and TSH of + 3.03 pmol/L (95% CI 2.64, 3.43) and - 0.03 mIU/L (95% CI - 0.25, 0.19), respectively. These differences are small compared to the width of the reference intervals. Thyroid-SPOT can predict the euthyroid set point remarkably well, especially for TSH with a 10-16-fold spread in magnitude between population normal limits. CONCLUSION: Every individual's equilibrium euthyroid set point is unique. Thyroid-SPOT serves as an accurate, precise and reliable targeting system for optimal personalized restoration of euthyroidism. This algorithm can guide clinicians in L-thyroxine dose titrations to resolve persistent dysthyroid symptoms among challenging cases harbouring "normal TFT" within the laboratory ranges but differing significantly from their actual euthyroid set points.

New insights on the mechanism(s) of the dominant negative effect of mutant thyroid hormone receptor in generalized resistance to thyroid hormone.

Generalized resistance to thyroid hormone (GRTH) is a syndrome of hyposensitivity to triiodothyronine (T3) that displays autosomal dominant inheritance. The genetic defect commonly lies in the ligand-binding domain of one of the TR beta alleles. Since there are two major thyroid hormone receptor (TR) isoforms, TR alpha and TR beta, it is not known how the mutant receptor mediates a dominant negative effect. Previously, we showed that T3 caused dissociation of TR homodimers and TR alpha/TR beta dimers from several thyroid hormone response elements (TREs). Hence, we used the electrophoretic mobility shift assay to compare the effect of T3 on the DNA binding of mutant TR beta-1 (Mf-1) from a kindred with GRTH with normal TR beta. Mf-1 bound better as a homodimer than TR beta, but dissociated from DNA only at high T3 concentrations. Both receptors heterodimerized with nuclear auxiliary proteins. They also dimerized with TR alpha and with each other. Surprisingly, T3 disrupted the DNA binding of the Mf-1/TR isoform dimers. Thus, mechanisms for the dominant negative effect by mutant TRs likely involve either increased binding to TREs by mutant homodimers that cannot bind T3 (hence cannot dissociate from DNA) and/or the formation of inactive mutant TR/nuclear protein heterodimers.

Differential modulation of gene induction by glucocorticoids and antiglucocorticoids in rat hepatoma tissue culture cells.

Studies of glucocorticoid and antiglucocorticoid induction of tyrosine aminotransferase (TAT) in two rat hepatoma cell lines (Fu5-5 and HTC) are described. These studies revealed several phenomena that are not consistent with the current models of steroid hormone action: (a) TAT induction occurred at glucocorticoid levels below those required for comparable receptor occupancy in Fu5-5, but not in HTC, cells; (b) the ability of antiglucocorticoids to induce TAT is higher in Fu5-5 than in HTC cells; (c) the values of the amount of TAT agonist activity with the antiglucocorticoid dexamethasone 21-mesylate and of log10 of the dexamethasone concentration required for half-maximal induction of TAT were not constant over time but varied in a linear, reciprocal manner. This modulation was seen for several glucocorticoids and antiglucocorticoids at the level of both TAT enzyme and mRNA but not for two other glucocorticoid inducible genes in the same cells. These results, plus the fact that a similar difference in the concentration required for half-maximal TAT induction in Fu5-5 cells was seen for both glucocorticoids and cyclic AMP, argue that the modulation occurs at some point distal to receptor-steroid complex binding to the biologically active nuclear sites but proximal to translation of TAT mRNA. In order to explain these results, it is pointed out that models involving second messengers are entirely appropriate for steroid hormone action. The participation of a modulated trans-acting factor in such a model may explain the above results.

New advances in understanding the molecular mechanisms of thyroid hormone action.

Thyroid hormone regulation o f gene transcription is a complex process. There are multiple thyroid hormone receptors (TRs) encoded on separate genes that bind to thyroid hormone-response elements (TREs) of target genes containing different orientation and spacing of half-sites. Additionally, there are multiple TR complexes-monomers, homodimers, and heterodimers with other related nuclear proteins-which bind to TREs and may play important roles in gene transcription. Recently, it has been shown that DNA binding of these TR complexes can be differentially regulated by either ligand or TR phosphorylation. Diversity among TR complexes and TREs, as well as mechanisms for regulating TR binding to TREs, may enable sensitive and precise transcriptional control of target genes.

Thyroid hormone regulation of hepatic genes in vivo detected by complementary DNA microarray.

The liver is an important target organ of thyroid hormone. However, only a limited number of hepatic target genes have been identified, and little is known about the pattern of their regulation by thyroid hormone. We used a quantitative fluorescent cDNA microarray to identify novel hepatic genes regulated by thyroid hormone. Fluorescent-labeled cDNA prepared from hepatic RNA of T3-treated and hypothyroid mice was hybridized to a cDNA microarray, representing 2225 different mouse genes, followed by computer analysis to compare relative changes in gene expression. Fifty five genes, 45 not previously known to be thyroid hormone-responsive genes, were found to be regulated by thyroid hormone. Among them, 14 were positively regulated by thyroid hormone, and unexpectedly, 41 were negatively regulated. The expression of 8 of these genes was confirmed by Northern blot analyses. Thyroid hormone affected gene expression for a diverse range of cellular pathways and functions, including gluconeogenesis, lipogenesis, insulin signaling, adenylate cyclase signaling, cell proliferation, and apoptosis. This is the first application of the microarray technique to study hormonal regulation of gene expression in vivo and should prove to be a powerful tool for future studies of hormone and drug action.

An inhibitory region of the DNA-binding domain of thyroid hormone receptor blocks hormone-dependent transactivation.

We have employed a chimeric receptor system in which we cotransfected yeast GAL4 DNA-binding domain/retinoid X receptor beta ligand-binding domain chimeric receptor (GAL4RXR), thyroid hormone receptor-beta (TRbeta), and upstream activating sequence-reporter plasmids into CV-1 cells to study repression, derepression, and transcriptional activation. In the absence of T3, unliganded TR repressed transcription to 20% of basal level, and in the presence of T3, liganded TRbeta derepressed transcription to basal level. Using this system and a battery of TRbeta mutants, we found that TRbeta/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, an AF-2 domain mutant (E457A) mediated basal repression but not derepression, suggesting that interaction with a putative coactivator at this site may be critical for derepression. Interestingly, a mutant containing only the TRbeta ligand binding domain (LBD) not only mediated derepression, but also stimulated transcriptional activation 10-fold higher than basal level. Studies using deletion and domain swap mutants localized an inhibitory region to the TRbeta DNA-binding domain. Titration studies further suggested that allosteric changes promoting interaction with coactivators may account for enhanced transcriptional activity by LBD. In summary, our findings suggest that TR heterodimer formation with RXR is important for repression and derepression, and coactivator interaction with the AF-2 domain may be needed for derepression in this chimeric system. Additionally, there may be an inhibitory region in the DNA-binding domain, which reduces TR interaction with coactivators, and prevents full-length wild-type TRbeta from achieving transcriptional activation above basal level in this chimeric receptor system.

Interactions of estrogen- and thyroid hormone receptors on a progesterone receptor estrogen response element (ERE) sequence: a comparison with the vitellogenin A2 consensus ERE.

The identification of hormone response elements in the promoter regions of hormonally regulated genes has revealed a striking similarity between the half-site of the estrogen-response element (ERE) and a consensus sequence constituting the thyroid hormone-response element. Because of the potential for thyroid hormone (T3) to affect estrogen (E)- and progesterone-dependent female reproductive behavior via EREs, we have begun to investigate the activity of an ERE identified in the progesterone receptor (PR) proximal promoter and its interactions with the estrogen receptor (ER) and thyroid hormone receptors (TR). In addition, we have compared ER and TR interactions on the PR ERE construct with that of the vitellogenin A2 (vit A2) consensus ERE. Electrophoretic mobility shift assays demonstrated that TR binds to the PR ERE as well as to the consensus ERE sequence in vitro. Further, these two EREs were differentially regulated by T3 in the presence of TR. T3 in the presence of TR alpha increased transcription from a PR ERE construct approximately 5-fold and had no inhibitory effect on E induction. Similarly, T3 also activated a beta-galactosidase reporter construct containing PR promoter sequences spanning -1400 to +700. In addition, the TR isoforms beta1 and beta2 also stimulated transcription from the PR ERE construct by 5- to 6-fold. A TR alpha mutant lacking the ability to bind AGGTCA sequences in vitro failed to activate transcription from the PR ERE construct, demonstrating dependence on DNA binding. In contrast to its actions on the PR ERE construct, TR alpha did not activate transcription from the vit A2 consensus ERE but rather attenuated E-mediated transcriptional activation. Attenuation from the vit A2 consensus ERE is not necessarily dependent on DNA binding as the TR alpha DNA binding mutant was still able to inhibit E-dependent transactivation. In contrast to TR alpha, the isoforms TRbeta1 and TRbeta2 failed to inhibit E-induced activation from the vit A2 consensus ERE. These results demonstrate that the PR ERE construct differs from the vit A2 consensus ERE in its ability to respond to TRs and that divergent pathways exist for activation and inhibition by TR. Since ERs, PRs, and TRs are all present in hypothalamic neurons, these findings may be significant for endocrine integration, which is important for reproductive behavior.

Region-specific antiglucocorticoid receptor antibodies selectively recognize the activated form of the ligand-occupied receptor and inhibit the binding of activated complexes to deoxyribonucleic acid.

A synthetic 18-amino acid peptide (Cys500-Lys517) was used to raise polyclonal antibodies in rabbits to the glucocorticoid receptor (GR). The sequence of this peptide is identical to that of residues 500-517 of the rat and 481-498 of the human GR. This sequence overlaps the carboxy-terminal end of the core DNA-binding domain and the amino-terminus of the hinge region of the receptor. Antiserum (AP64) was obtained which recognized both human and rat GR, as determined by immunoblots of receptors immunopurified with authentic anti-GR antibodies, immunoadsorption of both specific [3H]dexamethasone-bound GR and 98K receptors that were specifically covalently labeled by [3H]dexamethasone mesylate, and AP64-induced shifts in the elution position of monomeric [3H]dexamethasone-bound GR from Sephacryl S-300. The specificity of AP64 was demonstrated by the ability of the immunizing peptide, but not a peptide of similar length, to inhibit both the antibody-induced change in elution position from Sephacryl S-300 and the antibody-mediated immobilization of [3H]dexamethasone-bound complexes by protein-A. Further studies indicated that AP64 did not react with native steroid-free GR or with steroidbound (or affinity-labeled) unactivated GR, but did selectively associated with monomeric activated, steroid-bound (or affinity labeled) complexes. AP64 also inhibited the DNA binding of activated complexes in a manner that was specifically blocked by the immunizing peptide. Collectively, these data allow the direct localization of a structural region of the GR that is occluded in the unactivated complex but exposed as a result of activation.

The glucocorticoid receptor interacting protein 1 (GRIP1) localizes in discrete nuclear foci that associate with ND10 bodies and are enriched in components of the 26S proteasome.

The glucocorticoid receptor interacting protein-1 (GRIP1) is a member of the steroid receptor coactivator (SRC) family of transcriptional regulators. Green fluorescent protein (GFP) fusions were made to full-length GRIP1, and a series of GRIP1 mutants lacking the defined regulatory regions and the intracellular distribution of these proteins was studied in HeLa cells. The distribution of GRIP1 was complex, ranging from diffuse nucleoplasmic to discrete intranuclear foci. Formation of these foci was dependent on the C-terminal region of GRIP1, which contains the two characterized transcriptional activation domains, AD1 and AD2. A subpopulation of GRIP1 foci associate with ND10s, small nuclear bodies that contain several proteins including PML, SP100, DAXX, and CREB-binding protein (CBP). Association with the ND10s is dependent on the AD1 of GRIP1, a region of the protein previously described as a CBP-interacting domain. The GRIP1 foci are enriched in components of the 26S proteasome, including the core 20S proteasome, PA28alpha, and ubiquitin. In addition, the irreversible proteasome inhibitor lactacystin induced an increase in the total fluorescence intensity of the GFP-GRIP1 expressing cells, demonstrating that GRIP1 is degraded by the proteasome. These findings suggest the intriguing possibility that degradation of GRIP1 by the 26S proteasome may be a key component of its regulation.

Mutant and wild-type androgen receptors exhibit cross-talk on androgen-, glucocorticoid-, and progesterone-mediated transcription.

Androgen, glucocorticoid, and progesterone receptors (ARs, GRs, and PRs) often can regulate transcription via composite hormone response elements in target genes. We have used artificial and natural mutant ARs from patients with androgen resistance to study their effects on dominant negative activity on wild type AR, GR, and PR function on mouse mammary tumor virus (MMTV) and tyrosine aminotransferase (TAT) promoters. Artificial ARs that contained internal deletions within the amino-terminal region had minimal transcriptional activity but blocked ligand-mediated transcription by wild type AR. Mutants containing deletions of the DNA-binding and ligand-binding domains had minimal or weak dominant negative activity. We then tested the ability of wild type and mutant ARs to modulate GR- and PR-mediated transcriptional activity. The amino-terminal deletion mutants exerted dominant negative effects on GR- and PR-mediated activity, both in the absence and presence of testosterone. Surprisingly, wild type AR, which had approximately 20% of the maximal transcriptional activity of GR on the MMTV promoter, also had dominant negative activity on dexamethasone-regulated transcription mediated by GR. This dominant negative activity likely involves DNA binding because a point mutation in the DNA-binding domain abrogated such activity of an amino-terminal deletion mutant. Additionally, natural human AR mutants from patients with androgen resistance, which do not bind either DNA or ligand, did not block dexamethasone-mediated transcription. In summary, these studies suggest that mutant and wild type ARs can display dominant negative activity on other steroid hormone receptors that bind to a composite hormone response element This cross-regulation may be important in regulating maximal transcriptional activity in tissues where these receptors are coexpressed and may contribute to the phenotype of patients with steroid hormone resistance.

Aberrant alternative splicing of thyroid hormone receptor in a TSH-secreting pituitary tumor is a mechanism for hormone resistance.

Patients with TSH-secreting pituitary tumors (TSHomas) have high serum TSH levels despite elevated thyroid hormone levels. The mechanism for this defect in the negative regulation of TSH secretion is not known. We performed RT-PCR to detect mutations in TRbeta from a surgically resected TSHoma. Analyses of the RT-PCR products revealed a 135-bp deletion within the sixth exon that encodes the ligand-binding domain of TRbeta2. This deletion was caused by alternative splicing of TRbeta2 mRNA, as near-consensus splice sequences were found at the junction site and no deletion or mutations were detected in the tumoral genomic DNA. This TRbeta variant (TRbeta2spl) lacked thyroid hormone binding and had impaired T3-dependent negative regulation of both TSHbeta and glycoprotein hormone alpha-subunit genes in cotransfection studies. Furthermore, TRbeta2spl showed dominant negative activity against the wild-type TRbeta2. These findings strongly suggest that aberrant alternative splicing of TRbeta2 mRNA generated an abnormal TR protein that accounted for the defective negative regulation of TSH in the TSHoma. This is the first example of aberrant alternative splicing of a nuclear hormone receptor causing hormonal dysregulation. This novel posttranscriptional mechanism for generating abnormal receptors may occur in other hormone-resistant states or tumors in which no receptor mutation is detected in genomic DNA.

Short chain fatty acids increase prolactin and growth hormone production and alter cell morphology in the GH3 strain of rat pituitary cells.

Treatment of GH3 cells for 24-72 h with sodium valerate (1 mM) increased 2- to 4-fold the production of both PRL and GH. There was a concomitant change in the morphology of the cells that resembled that produced by TRH and epidermal growth factor. The increases in hormone production and changes in morphology were reversible. The sodium salts of butyric, hexanoic, caprylic, nonanoic, and dodecanoic acids also increased hormone production and altered cell morphology, whereas the salts of formic, acetic, and isobutyric acids did not. Acute (1-h) treatment with the fatty acids, unlike the effects of TRH and epidermal growth factor, did not increase the release of PRL from the cells.

9-cis retinoic acid regulation of rat growth hormone gene expression: potential roles of multiple nuclear hormone receptors.

Rat GH (rGH) gene expression is increased by both thyroid hormone (T3) and all-trans retinoic acid (RA) via a composite hormone response element (HRE) containing three putative half-sites (rGH-HRE). However, it is not known whether 9-cis RA (9cRA) also can regulate rGH gene expression. In this study, we performed a Northern blot analysis that demonstrated that 9cRA, as well as T3 and RA, increased rGH messenger RNA expression in rat pituitary GH3 cells. Transient transfection studies in GH3 cells, using reporter plasmids containing the rGH-HRE and mutated half-sites, revealed that 9cRA-stimulation of rGH transcription was mediated by the rGH-HRE and that all three half-sites are necessary. Additionally, we performed cotransfection studies to elucidate the particular receptor complexes involved in 9cRA regulation of rGH gene expression using CV-1 cells, which contain little or no endogenous RA receptors, and thyroid hormone receptors. Interestingly, in the presence of either retinoid X receptor alone, RA receptors alone, or both receptors, 9cRA caused similar induction of transcriptional activity. However, cotransfection of thyroid hormone receptors with these receptors repressed basal and blocked 9cRA-induced transcriptional activity in the absence of T3. Our data suggest that 9cRA-stimulation of rGH transcription is likely mediated by 9cRA-bound retinoid X receptor- and/or RA receptor-containing complexes but not by thyroid hormone receptor-containing complexes. Our studies provide evidence that several different members of the nuclear hormone receptor family can interact on this composite DNA element, with transcription stimulated or blocked depending on the presence or absence of cognate ligands.

Characterization and tissue expression of multiple triiodothyronine receptor-auxiliary proteins and their relationship to the retinoid X-receptors.

Thyroid hormone receptor (TR) binding to thyroid hormone response elements is enhanced by heterodimerization with T3 receptor auxiliary proteins (TRAPs). Although retinoid X-receptors (RXRs) behave similarly to TRAP by heterodimerizing with TRs and enhancing TR binding to thyroid hormone response elements, it is not known whether endogenous TRAPs are RXRs. In this study, we used the electrophoretic mobility shift assay to demonstrate that at least two different TR-TRAP heterodimer complexes can be formed from nuclear extracts of various rat tissues and cell lines. Additionally, the TRAPs appear to be differentially expressed in rat tissues. In antibody supershift experiments, most of the faster migrating TR-TRAP heterodimer bands and some of the slower migrating TR-TRAP heterodimer bands were recognized in several tissues and cell lines by the anti-RXR alpha-specific antibody. Immunodepletion assays showed that the slower migrating TR-TRAP heterodimer bands in most tissues and cell lines were recognized by the anti-RXR beta-specific antibody. Therefore, we have demonstrated that RXR alpha and RXR beta are endogenous TRAPs in a variety of tissues and cell lines, and are differentially expressed. We speculate that this heterogeneity of TRAP distribution may contribute to tissue and cell-specific expression of T3-regulated genes.

Basal and thyroid hormone receptor auxiliary protein-enhanced binding of thyroid hormone receptor isoforms to native thyroid hormone response elements.

There are three known isoforms of the rat thyroid hormone receptor, TR alpha-1, TR beta-1, and TR beta-2. The first two are expressed in all tissues, whereas TR beta-2 appears to be expressed only in the pituitary. The differences in the roles of the three receptor isoforms are unknown, but may involve preferential interaction with different subsets of thyroid hormone-regulated genes in different tissues. We tested the binding of the three TR isoforms to putative thyroid hormone response elements (TREs) from genes that are expressed in the pituitary or other tissues and are regulated by thyroid hormone. In vitro translated 35S-labeled rat TR alpha-1, rat TR beta-2, and human TR beta-1 receptors were bound to a battery of biotinylated synthetic deoxyribonucleotides containing naturally occurring putative TREs from genes expressed either in only pituitary (rat glycoprotein hormone alpha-subunit, TSH beta-subunit, and GH) or in nonpituitary (rat alpha-myosin heavy chain, malic enzyme, and Moloney murine leukemia virus promoter) tissues. All three receptor forms bound to each of the TREs. TR beta-2 did not show preferential binding to TREs of pituitary-specific genes compared to TR beta-1. Additionally, TR alpha-1 had a similar TRE-binding pattern as the TR beta s, except for possibly less binding to rat glycoprotein hormone alpha-subunit TRE. Finally, rat pituitary and liver nuclear extracts enhanced TR binding to TREs, with the greatest enhancement seen with the alpha-subunit TRE. These studies suggest that all TR isoforms bind similarly to native TREs. Also, TR binding to TREs can be differentially enhanced by interactions with nuclear proteins.

Steroid hormone receptors selectively affect transcriptional activation but not basal repression by thyroid hormone receptors.

Thyroid hormone receptors (TRs) and steroid hormone receptors belong to a large superfamily of nuclear hormone receptors. The interactions between these receptor subfamilies are poorly understood. In this study, cotransfection assays were used to examine the effects of estrogen and glucocorticoid receptors on TR-mediated repression of basal transcription by unliganded TR and transcriptional activation by liganded TR with two different thyroid hormone response element-containing reporter plasmids. Surprisingly, it was found that steroid hormone receptors blocked T3-mediated transcriptional activation with little or no effect on basal repression by unliganded TR. The mechanism for blocking TR-mediated transcriptional activation does not require steroid hormone receptor binding to the thyroid hormone response element but, rather, may involve titration of a critical coactivator(s) required for T3-mediated transcriptional activation. These studies strongly suggest divergent pathways for transcriptional activation and basal repression by TRs. Additionally, these studies raise the potential for nuclear hormone receptors to modulate TR-mediated transcriptional activation in steroid hormone-responsive tissues.

Expression and hormonal regulation of coactivator and corepressor genes.

Steroid/thyroid/retinoid receptors are members of the nuclear receptor superfamily and ligand-inducible transcription factors. These receptors modulate transcription of various cellular genes, either positively or negatively, by interacting with specific hormone-response elements located in the target gene promoters. Recent data show that nuclear receptors enhance or inhibit transcription by recruiting an array of coactivator and corepressor proteins to the transcription complex. We examined and compared the expression of four coactivator (steroid receptor coactivator-1 and E1A-associated 300-kDa protein) and corepressor (SMRT and N-CoR) genes in a number of tissues including several endocrine glands and cell lines. We also addressed whether their messenger RNA levels are hormonally regulated by studying the effects of thyroid hormone (T3) and estrogen (E2) treatment in rat pituitary cells (GH3) in vitro and in anterior pituitary in vivo. Our studies show that there are distinct tissue-specific expression patterns of these genes. We show that T3 and E2 regulate the expression of steroid receptor coactivator-1 messenger RNA in the anterior pituitary in addition to a gender-related difference. These tissue variations may have physiological implications for heterogeneity of hormone responses that are observed in normal and malignant tissues.

Anti-estrogenic compounds increase prolactin and growth hormone synthesis in clonal strains of rat pituitary cells.

Established clonal strains of rat pituitary cells, GH-cells, responded prior to 1974 to 10(-11) to 10(-8)M 17beta-estradiol by increasing prolactin synthesis 2-fold and decreasing the production of growth hormone to between 20 and 70% of control values. In experiments in 1975-76, these effects of estradiol were no longer statistically significant. Unexpectedly, the estrogen antagonists enclomiphene, CI-628, tamoxifen, and Lilly 88751 caused increases in both prolactin and growth hormone synthesis at concentrations of about 10(-7) to 10(-6)M after one week of treatment. These increases were prevented by 10(-8)M 17beta-estradiol. Prolactin synthesis remained elevated for at least 11 days after removal of enclomiphene from the culture medium but, in the presence of estradiol, synthesis approached control levels by 11 days. In GH-cells, compounds which are estrogen antagonists in other systems mimic the previously observed effect of estradiol on prolactin synthesis, but have an effect opposite to that of estradiol on growth hormone, namely stimulation of its synthesis.

Isoform-specific retinoid-X receptor (RXR) antibodies detect differential expression of RXR proteins in the pituitary gland.

There are three known isoforms of the retinoid-X receptor (RXR): RXR alpha, RXR beta, and RXR gamma. RXR alpha and RXR beta messenger RNAs are widely expressed, whereas RXR gamma messenger RNA is restricted to only a few tissues, including embryonic pituitary gland. Little is known about the level of expression and cell distribution of RXR proteins in the adult pituitary gland. To examine these issues further, we raised isoform-specific polyclonal antibodies against each of the known mouse RXR isoforms using synthetic peptides containing isoform-specific epitopes from the amino-terminal region. The specificity of each antibody was confirmed by immunoprecipitation, Western immunoblot analysis, and electrophoretic mobility shift assay with supershift studies of in vitro translated RXR isoforms. Immunocytochemical analysis showed that anti-RXR alpha and anti-RXR beta antisera stained the nuclei of most pituitary cells. In contrast, anti-RXR gamma antiserum stained the nuclei of only a few cells throughout the pituitary. In the hypothyroid state, however, a marked increase in both the number and density of RXR gamma-immunostained nuclei were observed compared to those in the euthyroid state. Double immunostaining studies of hypothyroid rat pituitary with antibodies against pituitary hormones indicated that RXR gamma protein was predominantly expressed in thyrotropes. Antibody supershift experiments using nuclear extracts of adult rat whole pituitary and rodent pituitary cell lines showed that anti-RXR gamma antibody could alter the mobility of protein-DNA complexes formed only from nuclear extracts of rat whole pituitary and thyrotropic TtT-97 cells. In contrast, anti-RXR alpha and anti-RXR beta antibodies could supershift protein-DNA complexes formed from nuclear extracts of all cell lines tested. RXR gamma protein expression in TtT-97 cells also was observed by Western immunoblot analyses. Therefore, there is thyrotrope-predominant expression of RXR gamma protein. We speculate that RXR gamma may play a role in the regulation of thyroid hormone target genes in thyrotropes and possibly cell type differentiation in the pituitary.

Heterodimerization and deoxyribonucleic acid-binding properties of a retinoid X receptor-related factor.

The extent thyroid hormone receptors (TRs) bind to AGGTCA-related motifs as monomers and/or homodimers, and as heterodimers with retinoid X receptors (RXRs) depends on the number, spacing, and orientation of these half-sites. Here we show that recombinant RXR alpha affects TR binding to DNA in diverse ways; it enhances recombinant TR beta 1 binding to four-nucleotide-spaced direct repeat and palindromes but not to inverted palindromes. We also used an endogenous factor termed RXR alpha-RF that cross-reacted with antibodies to RXR alpha and copurified and formed heterodimers on DNA with rat liver TRs (mostly TR beta 1 isoform), supporting the fact that endogenous TRs are commonly heterodimers. RXR alpha-RF formed, like recombinant RXR alpha, heterodimers on DNA with vitamin D and retinoic acid but not estrogen receptors. RXR alpha-RF differed from recombinant RXR alpha in that it provoked enhancement of TR beta 1 binding to DNA irrespective of half-site architecture, was resistant to heating to 50 C, and did not form heterodimers with recombinant TR alpha 2 on four-nucleotide-spaced direct repeat. The overall enhancement of TR-DNA recognition by endogenous RXR alpha-RF, not found in studies with recombinant RXR alpha, might exemplify properties acquired in vivo by endogenous RXRs; this could promote wider DNA recognition by TRs and expand the thyroid hormone transcriptional influence in the cell.