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To study the role of the thyroid hormone (TH) in cerebellar development, we generated transgenic mice expressing a dominant-negative TH receptor (TR) in cerebellar Purkinje cells. A mutant human TRβ1 (G345R), which binds to the TH-response element but cannot bind to T3, was subcloned into exon 4 of the full-length L7/Pcp-2 gene, which is specifically expressed in Purkinje and retinal rod bipolar cells. The transgene was specifically expressed in Purkinje cells in the postnatal cerebellum. Purkinje cell dendrite arborization was significantly delayed in the transgenic mice. Surprisingly, granule cell migration was also significantly delayed. In the primary cerebellar culture, TH-induced Purkinje cell dendrite arborization was also suppressed. In quantitative real-time RT-PCR analysis, the expression levels of several TH-responsive genes were altered. The expression levels of inositol trisphosphate receptor type 1 and retinoic acid receptor-related orphan receptorα mRNAs, which are mainly expressed in Purkinje cells, and brain-derived neurotrophic factor mRNA, which is expressed in both Purkinje and granule cells, were significantly decreased. The expression levels of neurotrophin-3 and hairless mRNAs, which are mainly expressed in granule cells, and myelin basic protein mRNA, which is mainly expressed in oligodendrocytes, were also decreased. The motor coordination of transgenic mice was significantly disrupted. These results indicate that TH action through its binding to TR in Purkinje cells is required for the normal cerebellar development. TH action through TR in Purkinje cells is also important for the development of other subsets of cerebellar cells such as granule cells and oligodendrocytes.

Steroid receptor coactivator-1 (SRC-1) plays a crucial role in nuclear receptor-mediated transcription including thyroid hormone receptor (TR)-dependent gene expression. Interaction of the TR-ligand binding domain and SRC-1 through LXXLL motifs is required for this action. However, potential interactions between the TRbeta1-N terminus (N) and SRC-1 have not been explored and thus are examined in this manuscript. Far-Western studies showed that protein construct containing TRbeta1-N + DNA binding domain (DBD) bound to nuclear receptor binding domain (NBD)-1 (amino acid residue, aa 595-780) of SRC-1 without ligand. Mammalian two-hybrid studies showed that NBD-1, as well as SRC-1 (aa 595-1440), bound to TRbeta1-N+DBD in the absence of ligand in CV-1 cells. However, NBD-2 (aa 1237-1440) did not bind to this protein. Glutathione-S-transferase pull-down studies showed that TRbeta1-N (aa 1-105) bound to the broad region of SRC-1-C terminus. Expression vectors encoding a series of truncations and/or point mutations of TRbeta1 were used in transient transfection-based reporter assays in CV-1 cells. N-terminal truncated TRbeta1 (DeltaN-TRbeta1) showed lower activity than that of wild-type in both artificial F2-thyroid hormone response element and native malic enzyme response element. These results suggest that there is the interaction between N terminus of TRbeta1 and SRC-1, which may serve a full activation of SRC-1, together with activation function-2 on TRbeta1-mediated transcription.

The response of pituitary gonadotropes to GnRH correlates directly with the concentration of GnRH receptors (GnRHRs) on the cell surface, which is mediated in part at the level of GnRHR gene expression. We have previously localized GnRH responsiveness in the mouse GnRHR (mGnRHR) gene promoter to two elements: activating protein-1 and sequence underlying responsiveness to GnRH-1. This study was designed to investigate potential synergy between GnRH and activin A in transcriptional activation of the mGnRHR gene. In functional transfection studies using alphaT3-1 cells, GnRH agonist stimulation of the mGnRHR gene promoter (-765/+62) resulted in a 10.9-fold increase in activity, which was further increased 2-fold (to 21.3-fold) following activin pretreatment. Activin pretreatment alone had no effect. Deletion of region -387/-308 or mutation of a putative SMAD-binding element at -331/-324 (5'-GTCTAG[T]C-3') abrogated the augmented response to GnRH in the presence of activin but not the response to GnRH alone. Overexpression of SMAD2 and SMAD3 along with SMAD4 increased transcriptional activity of the mGnRHR gene, which was further increased by GnRH agonist stimulation. These data demonstrate that activin augments GnRH-mediated transcriptional activation of the mGnRHR gene and suggest that this effect may be mediated through SMAD transcription factors.

Thyroid hormone receptor (TR), a ligand-mediated transcription factor, binds to a DNA sequence known as a thyroid-hormone response element (TRE) to activate or repress transcription of target genes. Recently, studies have shown that Ca2+/calmodulin-dependent protein kinases (CaMKs) may be involved in regulating gene transcription via phosphorylation of specific transcription factors, including RORalpha, a retinoic acid-related orphan nuclear hormone receptor. In this light, we examined the effect of CaMK type IV (CaMKIV) and RORalpha, which also shown to influence thyroid hormone action, on TR-mediated transcription using a transient transfection assay. Expression vectors containing TR, vitamin D receptor (VDR), and estrogen receptor (ER) were cotransfected in CV-1 cells with RORalpha and/or constitutively active CaMKIV and thymidine kinase promotor-luciferase reporter vector containing their cognate response elements. When CaMKIV or RORalpha was co-transfected with TR, the T3-induced transcription was significantly augmented compared to that induced by TR alone. When both were co-transfected with TR, T3-induced transcription was augmented additively. In contrast, the augmentation by CaMKIV or ROR on ligand-induced transcription was not detected with VDR and ER. Hence, these results indicate that the augmentation mediated by CaMKIV and RORalpha is specific for TR-mediated transcription on TRE. Our results suggest that CaMKIV, as well as RORalpha, play important roles in TR-mediated transcription on TREs.

Thyroid hormone (TH) plays an important role in the postnatal development of the rodent cerebellum, particularly within the first 2 weeks of postnatal life. This action is exerted through the regulation of specific genes during development and is mediated by coactivator and corepressor proteins that determine transcriptional repression or activation, respectively. Thus, we hypothesized that the effect of TH on rodent cerebellar development could be influenced by the relative amounts of coactivator and corepressor proteins in vivo. These ratios might be modulated in an age-specific manner and/or by hormones to generate the "critical period" of TH action. To examine this hypothesis, we cloned rat complementary DNA fragments corresponding to coactivators (SRC1, TIF2 and TRAM1) and corepressors (N-CoR and SMRT), and studied the ontogenic changes in their corresponding messenger RNAs in rat cerebellum of normal and hypothyroid rats during postnatal development, using a RNase protection assay. We found an increased expression of SRC1 and TIF2, as well as of N-CoR, during rat cerebellar development but no change in the expression of SMRT and TRAM1 genes. However, thyroid hormone status did not affect the expression of coactivator and corepressor genes in the cerebellum. These results indicate that coactivator and corepressor messenger RNAs exhibit differential expression through cerebellar development but are not regulated by TH during this period.

Thyroid hormone (TH) plays a critical role in normal cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. This action could be exerted in part through brain-derived neurotropic factor (BDNF), as cerebellar BDNF messenger RNA (mRNA) expression is lower, and replacement of BDNF partially reverses the abnormal neurogenesis in the hypothyroid rat. The rat BDNF gene consists of four noncoding exons (exons I–IV), each of which is linked to a different promoter, and a protein-coding exon (exon V). To study promoter-specific regulation of the BDNF gene by TH, ribonuclease protection assay of each exon mRNA was performed using total developing rat cerebellar RNA. During cerebellar development, all exon mRNAs were detected, but with different expression patterns; among noncoding exon mRNAs, exon II mRNA was the most abundant. Daily TH replacement induced a 3-fold increase in exon II mRNA on postnatal day (P) 15. On P30, exon II mRNA was still much greater in the TH-replaced animal. Exon I mRNA was detected on P2 and P7. However, in contrast to exon II mRNA, TH treatment suppressed the expression of exon I mRNA on P2. Exon III and IV mRNAs were not detected on P2 and P7, but small amounts were observed starting on P15 in TH-replaced animals. They were not detected by P30 in hypothyroid animals. In contrast, in the cerebral cortex, although all exons are differentially regulated during development, the expression of each mRNA was not significantly altered by TH. These results indicate that TH regulates BDNF gene expression in a promoter-, developmental stage-, and brain region-specific manner, which may play an important role in region- and stage-specific regulation of brain development by TH.

Ligand-dependent nuclear hormone receptors (NRs), such as retinoic acid and thyroid hormone receptors, play critical roles in diverse aspects of development. They enhance or repress transcription by recruiting an array of coactivator and corepressor proteins, which function as signaling intermediates between the NRs and the basal transcriptional machinery. To study the possible involvement of these cofactors on tissue-specific regulation of gene expression by NRs, we examined the expression of the coactivator SRC-1 mRNA during mouse embryogenesis by in situ hybridization (ISH). 35S-labeled riboprobe specific for SRC-1 mRNA was used for analysis. The distribution of this transcript was studied from 8.5 to 18.5 embryonic days (E8.5-E18.5) and in postnatal day 15 (P15). The SRC-1 transcript was largely ubiquitously expressed, even on E8.5. At E14.5 and E18.5, highest levels of SRC-1 transcript was found in the olfactory epithelium. Significant SRC-1 hybridization signal was also detected in the neocortex, anterior pituitary and heart. We conclude that (1) SRC-1 mRNA is widely expressed in the developing embryo, and (2) SRC-1 mRNA is expressed at the highest level in the olfactory epithelium, suggesting that this coactivator may be involved in the development and/or function of the olfactory system.

This study is designed to clarify the role of an orphan nuclear hormone receptor, ROR alpha, on thyroid hormone (TH) receptor (TR)-mediated transcription on a TH-response element (TRE). A transient transfection study using various TREs [i.e., F2 (chick lysozyme TRE), DR4 (direct repeat), and palindrome TRE] and TR and ROR alpha1 was performed. When ROR alpha1 and TR were cotransfected into CV1 cells, ROR alpha1 enhanced the transactivation by liganded-TR on all TREs tested without an effect on basal repression by unliganded TR. By electrophoretic mobility shift assay, on the other hand, although ROR alpha bound to all TREs tested as a monomer, no (or weak) TR and ROR alpha1 heterodimer formation was observed on various TREs except when a putative ROR-response element was present. The transactivation by ROR alpha1 on a ROR-response element, which does not contain a TRE, was not enhanced by TR. The effect of ROR alpha1 on the TREs is unique, because, whereas other nuclear hormone receptors (such as vitamin D receptor) may competitively bind to TRE to exert dominant negative function, ROR alpha1 augmented TR action. These results indicate that ROR alpha1 may modify the effect of liganded TR on TH-responsive genes. Because TR and ROR alpha are coexpressed in cerebellar Purkinje cells, and perinatal hypothyroid animals and ROR alpha-disrupted animals show similar abnormalities of this cell type, cross-talk between these two receptors may play a critical role in Purkinje cell differentiation.

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.

Deficiency of thyroid hormone (TH) during the perinatal period results in severe neurological abnormalities in rodent cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. Of note, a mutant mouse, staggerer, in which the orphan nuclear hormone receptor ROR alpha gene is disrupted, exhibits cerebellar abnormalities similar to those seen in the hypothyroid animals, despite normal thyroid function. We, therefore, speculated that TH (tetraiodo-L-thyronine; T4) may regulate ROR alpha gene expression, which then may regulate genes essential for normal brain development. To test this hypothesis, we studied the changes in ROR alpha gene expression in perinatal hypothyroid rat cerebellum and the effect of TH replacement using Northern blot analysis, ribonuclease protection assay and in situ hybridization histochemistry. During cerebellar development, an approximately 3-fold increase in the cerebellar content of ROR alpha messenger RNA (mRNA) was seen in both propylthiouracil-treated, and propylthiouracil-treated and T4-replaced animals. However, the increase was accelerated when T4 was injected, although the ROR alpha mRNA content was identical, with or without T4, by 30 days after birth (P30). In contrast, T4 treatment suppressed the TH receptor alpha1 and c-erbA alpha2 mRNA content by P30; retinoic acid X receptor-beta mRNA content was not influenced by thyroid status. A significant hybridization signal for ROR alpha mRNA was seen only over Purkinje cells in the cerebellar cortex by in situ hybridization histochemistry. These results indicate that TH alters the timing of expression of the ROR alpha gene in the Purkinje cells of the cerebellar cortex, which may, in turn, influence Purkinje cell differentiation.

The regulation of LH and FSH subunit gene expression is under the control of GnRH. Physiological changes in the frequency of pulsatile GnRH release from the hypothalamus result in differential stimulation of α-, LHβ-, and FSHβ-gene expression. Previous studies indicate that the GnRH receptor couples to G proteins of the Gq/11 family, with phosphoinositide turnover and its resultant increase in intracellular calcium concentration and protein kinase C (PKC) activation, to stimulate secretion of LH and FSH. However, the molecular mechanisms by which GnRH mediates its transcriptional effects remain largely unknown. We used GH3 cells, constitutively expressing the rat GnRH receptor (GGH3-1′ cells) and transiently transfected with a luciferase reporter gene controlled by either the α, LHβ, or FSHβ gene regulatory region (αLUC, LHβLUC, and FSHβLUC, respectively), to examine the roles of several signal transduction pathways in the GnRH-mediated stimulation of gonadotropin subunit gene expression. Activation of PK...

The hypothalamic hormone, GnRH, is released and transported to the anterior pituitary in a pulsatile manner, where it binds to specific high-affinity receptors and regulates gonadotropin biosynthesis and secretion. The frequency of GnRH pulses changes under various physiological conditions, and varying GnRH pulse frequencies have been shown to regulate differentially the secretion of LH and FSH and the expression of the gonadotropin alpha, LH beta, and FSH beta subunit genes in vivo. We demonstrate differential effects of varying GnRH pulse frequency in vitro in superfused primary monolayer cultures of rat pituitary cells. Cells were treated with 10 nM GnRH pulses for 24 h at a frequency of every 0.5, 1, 2, or 4 h. alpha, LH beta, and FSH beta messenger RNA (mRNA) levels were increased by GnRH at all pulse frequencies alpha and LH beta mRNA levels and LH secretion were stimulated to the greatest extent at a GnRH pulse frequency of every 30 min, whereas FSH beta mRNA levels and FSH secretion were stimulated maximally at a lower GnRH pulse frequency, every 2 h. GnRH receptor (GnRHR) mRNA levels also were increased by GnRH at all pulse frequencies and were stimulated maximally at a GnRH pulse frequency of every 30 min. Similar results were obtained when the dose of each pulse of GnRH was adjusted to maintain a constant total cumulative dose of GnRH over 24 h. These data show that gonadotropin subunit gene expression is regulated differentially by varying GnRH pulse frequencies in vitro, suggesting that the differential effects of varying GnRH pulse frequencies on gonadotropin subunit gene expression occur directly at the level of the pituitary. The pattern of regulation of GnRHR mRNA levels correlated with that of alpha and LH beta but was different from that of FSH beta. This suggests that alpha and LH beta mRNA levels are maximally stimulated when GnRHR levels are relatively high, whereas FSH beta mRNA levels are maximally stimulated at lower levels of GnRHR expression, and that the mechanism for differential regulation of the gonadotropins by varying pulse frequencies of GnRH may involve levels of GnRHR. Furthermore, these data suggest that the mechanisms whereby varying GnRH pulse frequencies stimulate alpha LH beta, and GnRHR gene expression are similar, whereas the stimulation of FSH beta mRNA levels may be different.

Two genes, c-ErbA alpha and c-ErbA beta, generate at least three functional T3 receptor (TR) isoforms in the rat: TR alpha-1, TR beta-1, and TR beta-2. The latter is an N-terminal splice variant of TR beta-1 whose expression is high in the pituitary gland, whereas the other isoforms are more widely expressed. It is believed that TR beta-2 might play an important role in the pituitary, but no specific biological activities have been defined. Using in vitro translated receptors, we were unable to detect striking isoform-specific differences in T3 binding, DNA binding, homodimerization and heterodimerization activities with retinoid X receptor in the electrophoretic mobility assay, or T3-dependent repression and activation of basal transcription in transfection assays. The N-terminus of TR beta is completely dispensible for these activities. However, we found that unliganded TR alpha-1 and TR beta-1, but not TR beta-2, can repress glucocorticoid receptor-mediated transcriptional activation if the reporter gene promoter contains binding sites for both receptor species. TR beta-2 also synergizes most efficiently with the glucocorticoid receptor in the presence for the of T3. The TR beta-2-specific N-terminus is required for these effects. This result indicates that the relative abundance of specific TR isoforms may determine the with the glucocorticoid receptor in the presence of T3. The TR beta-2 specific N-terminus is required for these effects. This result indicates that the relative abundance of specific TR isoforms may determine the quantitative response of a gene that is regulated by T3 and glucocorticoids. In particular, pituitary TR beta-2 may be an important mediator of GH gene expression, which is regulated by both of these hormones.

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.

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.

Four cell lines, stably transfected with rat GnRH receptor complementary DNA, have been prepared from the lactotropic GH3 cell line. All four lines (as well as the parent line and a line transfected with the vector DNA) show extensive rosettes of circular polyribosomes, characteristic of high protein synthetic activity, although secretory granules are virtually absent; the rough endoplasmic reticulum (rER) cisternae were short and straight. Instances were observed in which the ER reaches to the plasma membrane, suggesting a possible nongranular secretory route. All four lines (but not the parent or a control transfected line) expressed GnRH receptors that were down-regulated (1-5 h, depending on the cell line) after exposure to 10 nM GnRH; receptors then recovered (2-7 h). This pattern is reminiscent of the GnRH receptor in the primary gonadotrope cell cultures. All cell lines released PRL (4-96 h) in response to a GnRH agonist (D-tBuSer6-des-Gly10-Pro9-ethylamide-GnRH), an event that was inhibited by all three major classes of Ca+2 ion channel antagonists (methoxyverapamil, 1,4-dihydropyridines, and diltiazem); in contrast, GnRH-stimulated LH release from pituitary-derived primary cultures is only inhibited by methoxyverapamil. One line became refractory to GnRH analog stimulation after 24 h, although the other three released PRL vigorously up to the longest time point examined (96 h). All four lines responded substantially more robustly to 1 microgram/ml Buserelin than to 1 microgram/ml TRH. All four lines produced inositol phosphate metabolites and released immunoassayable cAMP (24 h) in response to treatment with Buserelin. These cell lines are good models for understanding the mechanisms by which the GnRH receptor is coupled to second messenger systems and for comparing these mechanisms with TRH-receptor coupling in the same cell.

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.

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.

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.

The recent isolation of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor (GnRHR) allows studies of the regulation of the synthesis of the GnRHR and its relationship to reproductive function. Analyses of the regulation of GnRHR mRNA levels in the rat pituitary in vivo revealed a progressive increase in levels to 2.0 +/- 0.2-fold after ovariectomy (OVX) and 5.2 +/- 1.3-fold after castration (CAST) (21 days post-operative), compared to intact adult female and male controls, respectively. Replacement therapy with 17 beta-estradiol benzoate in 21-day post-OVX female rats resulted in a marked decrease in GnRHR mRNA levels by 7 days, compared to controls. In contrast, therapy with testosterone propionate in 21-day post-CAST male rats resulted in only a modest decrease in GnRHR mRNA levels. Thus, manipulation of the reproductive endocrine system in vivo results in alterations in GnRHR synthesis at the pretranslational level, which parallel known changes in cell surface gonadotropin-releasing hormone (GnRH) binding activities. The treatment of superfused primary monolayer cultures of rat pituitary cells with hourly pulses of GnRH (10 nM, 6 min/h) resulted in a marked increase in GnRHR mRNA levels (12.8 +/- 4.3-fold compared to untreated cells). In contrast, treatment of cultured cells with continuous GnRH caused no change in GnRHR mRNA levels. These in vitro data show homologous regulation of GnRHR gene expression by GnRH, and suggest that the changes in GnRHR gene expression observed in vivo may be attributable at least in part to changes in the pattern of hypothalamic GnRH secretion.

In addition to its well known actions in stimulating TSH and PRL synthesis and secretion, TRH has been shown to decrease the concentration of thyroid hormone receptors (TRs) in GH4C1 cells as measured by nuclear thyroid hormone (T3) binding. In the present study we have investigated the effects of TRH on the levels of mRNA encoding the different forms of TR, TR beta-1, TR beta-2, and TR alpha-1 as well as that of the non-T3-binding variant, c-erbA alpha-2. GH3 cells were incubated with 100 nM TRH in the presence or absence of 1 nM T3 for 48 h, and mRNA levels were determined by Northern blot analysis. Results revealed that there is differential regulation of the individual TRs by TRH at the pretranslational level. The mRNA for the pituitary-specific form of TR, TR beta-2, was down-regulated by 60% by TRH in GH3 cells, while that of its alternative splice product, TR beta-1, was unchanged. A modest change was observed in TR alpha-1 mRNA levels, which were down-regulated by 20%; there was no change in c-erbA alpha-2 mRNA levels. Levels of nuclear T3 binding were assessed under the same conditions, and 100 nM TRH was found to decrease binding by 40% from 0.78 to 0.46 fmol/micrograms DNA. A similar change in nuclear T3 binding was seen after incubation with 1 nM T3. The effect of TRH on the GH mRNA response to T3 was investigated. In the absence of TRH there was a 4-fold induction of GH mRNA after incubation with 1 nM T3. In the presence of 100 nM TRH, no significant induction in GH mRNA by T3 was seen, indicating that T3 responsiveness as well as receptor concentration are diminished by TRH under these conditions.

Vitamin D receptor (VDR) is a ligand-dependent transcription factor that regulates target gene expression. Although VDR forms stable heterodimer complex with retinoid X receptors (RXRs) on vitamin D-response elements (VDREs), it is still not clear whether VDR/RXR heterodimers are the only VDR complexes responsible for vitamin D-mediated gene transcription. In this report, we analyzed the effect of nuclear receptor coactivators (SRC-1 and TRAM-1) on VDR homodimer and VDR/RXR heterodimer formation by electrophoretic mobility shift assay. We found that VDR forms stable homodimers after interaction with the coactivators on a VDRE (DR+3). Of particular note, DR+4 and DR+5 hormone-response elements (HREs) may also support such interactions. Cotransfection experiments revealed further that the coactivators enhance ligand-induced VDR transcription on these elements. Our studies suggest the important role of VDR homodimers, in addition to VDR/RXR heterodimers, in vitamin D-induced transactivation. Thus, specific coactivator-VDR interactions on HREs may determine target gene transactivation.

Our previous work has shown that 1.2 kb of the 5′ flanking region of the mouse GnRH receptor (mGnRH-R) gene is sufficient to direct tissue-specific expression in vitro. In this study, we have used the cell-specific regulatory sequences of the mGnRH-R gene promoter to target the expression of the simian virus 40 virus T antigen (TAg) to the pituitary gland of transgenic mice. A hybrid transgene, GnRH-R/TAg, was prepared using the −1164/+52 region of the mGnRH-R gene and +2533/+5234 sequences encoding the large T antigen of the simian virus 40. Two founders developed tumors of apparent pituitary origin at 44 (M28, female) and 50 (M25, male) days of age. M28 and M25 mice were about 50% underweight, and their gonads were grossly underdeveloped compared with wild-type litter mates. A third male founder, M29, developed a tumor at a later time (109 days). M29 was able to breed successfully and stably transmit the GnRH-R/TAg transgene. Mice of the M29 transgene line developed tumors at 4–5 months of age. Gross examination showed that the tumors extend from the sella and infiltrate into the inferior surface of the brain. In small tumors collected from young transgenic animals, normal pituitary cells as well as transition areas of increasing cellular atypia are evident. Frankly malignant cells are seen in all tumors. The pituitary tumors express the α-, FSHβ-, and LHβ-subunits and the GnRH-R messenger RNA, all markers of a gonadotrope but not of other anterior pituitary cell lineages. In summary, our studies indicate that 1.2 kb of the 5′-flanking region of the mGnRH-R gene can be used to target expression specifically to the gonadotropes of the pituitary gland in transgenic mice. The GnRH-R gene promoter-directed expression appears to be cell-specific and results in the formation of tumors that are primarily of gonadotropic origin.

GnRH plays a critical role in reproductive development and function by regulating the biosynthesis and secretion of the pituitary gonadotropins, LH and FSH. Although it is known that GnRH induces gonadotropin subunit gene transcription, the mechanism by which this occurs has not been elucidated. Studies have been hindered by the lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have transfected the rat pituitary GH3 cell line with the rat GnRH receptor complementary DNA. These cells, when cotransfected with regulatory regions of the LH or FSH subunit genes fused to a luciferase reporter gene, respond to GnRH with an increase in promoter activity comparable to that seen in primary rat pituitary cells. In this study, we have used this cell model to identify cis-acting elements of the LHβ gene that mediate stimulation by GnRH. Analysis of a series of 5′-deletion and internal deletion constructs has revealed two regions of the rat LHβ gene promoter involved in mediating the response to GnRH, region A (−490/−352) and region B (−207/−82). Fusion of region A upstream of a heterologous minimal promoter linked to the luciferase gene conferred GnRH responsiveness to the promoter, whereas region B did not. However, the presence of both regions A and B conferred a greater GnRH response than region A alone. Electrophoretic mobility shift assay revealed the presence of a protein(s) binding to region A using GH3 as well as αT3–1 nuclear extracts. Thus, region A (−490/−352) confers GnRH responsiveness to the LHβ subunit gene and binds to a protein(s) present in pituitary cell lines. DNA sequences in region B (−207/−82) also contribute to GnRH responsiveness. The identification of putative GnRH response elements in the rat LHβ gene promoter will aid in elucidation of the mechanisms of regulation of gene expression by GnRH.

The conserved diversity, restricted distribution, and differential regulation of the thyroid hormone receptor (TR) isoforms raise the possibility of isoform-specific functions. We have addressed the roles of individual TRs in GH gene expression in GH3 cells by using an isoform-specific antisense RNA to delete TRbeta1. An antisense RNA vector, directed against the isoform-specific coding sequence of the parent TRbeta1 complementary DNA, was constructed. Stable transfected GH3-derived cell lines expressing this construct were established. Appropriate control cell lines were established in parallel. Depletion of TRbeta1 in cells expressing the antisense construct was confirmed at both the messenger RNA and protein levels. Total TR expression was maintained in these cells by a reciprocal increase in TRbeta2 levels. This perturbation of the TR population was associated with a 10.5-fold increase in basal and a 5.0-fold increase in T3-stimulated GH gene expression, but no increase in total T3 binding of nuclear extracts. In transient cotransfection experiments, there were no differences between control cells and those expressing the antisense construct in either basal or T3-stimulated expression of reporters containing a variety of thyroid hormone response elements. Depletion of TRbeta1 in GH3 cells results in a reciprocal increase in TRbeta2. These changes are associated with increased basal and T3-stimulated GH gene expression, which are not due to a nonspecific enhancement of basal or hormone-stimulated transcription. We demonstrate that TRbeta1 is not required for T3 induction of the GH gene in GH3 cells and that TRbeta1 and TRbeta2 are not equivalent in their effects on basal repression of the GH promoter. The data illustrate the potential for isoform- and promoter-specific dissociation of the repression and activation properties of the TRs.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily implicated in adipocyte differentiation. The observations that PPAR alpha is a regulator of hepatic lipid metabolism and that the insulin-sensitizing thiazolidinediones are ligands for PPAR gamma suggest that cross-talk might exist between insulin signaling and PPAR activity, possibly through insulin-induced PPAR phosphorylation. Immunoprecipitation of endogenous PPAR alpha from primary rat adipocytes prelabeled with [32P]-orthophosphate and pretreated for 2 h with vanadate and okadaic acid demonstrated for the first time that PPAR alpha is a phosphoprotein in vivo. Treatment with insulin induced a time-dependent increase in PPAR phosphorylation showing a 3-fold increase after 30 min. Insulin also increased the phosphorylation of human PPAR alpha expressed in CV-1 cells. These changes in phosphorylation were paralleled by enhanced transcriptional activity of PPAR alpha and gamma. Transfection studies in CV-1 cells and HepG2 cells revealed a nearly 2-fold increase of PPAR activity in the presence of insulin. In contrast, insulin had no effect on the transcriptional activity of transfected thyroid hormone receptor in CV-1 cells, suggesting a PPAR-specific effect. Thus, insulin stimulates PPAR alpha phosphorylation and enhances the transcriptional activity of PPAR, suggesting that the transcriptional activity of this nuclear hormone receptor might be modulated by insulin-mediated phosphorylation.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. The conserved carboxy-terminal region of the ligand-binding domain (AF-2) has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivator(s). Using bacterially-expressed TR as a probe, far-Western-based expression cDNA library screening identified cDNAs that encode, in part, the recently reported partial steroid receptor coactivator-1 (SRC-1) sequence. Additional work, including 5' RACE, has characterized a full-length cDNA that encodes a approximately 160 kD protein as a putative thyroid hormone receptor coactivator (F-SRC-1). In vitro binding studies show that F-SRC-1 binds to a variety of nuclear hormone receptors in a ligand-dependent manner, along with TBP and TFIIB, suggesting that F-SRC-1 may play a role as a bridging molecule between nuclear hormone receptors and general transcription factors. Interestingly, AF-2 mutants also retain ligand-dependent interaction with F-SRC-1. Although F-SRC-1 recognizes the ligand-induced conformational changes of nuclear hormone receptors, our observations suggest that F-SRC-1 may bind directly with subregion(s) in nuclear hormone receptors other than the AF-2 region.

The formation of heterodimers with thyroid hormone receptor (TR) and other nuclear hormone receptors on thyroid hormone response elements (TREs) is important for T3-regulated gene transcription. The potential roles of retinoid X receptor-alpha (RXR alpha) and apolipoprotein regulatory protein-1 (ARP-1) in T3-mediated transcription were studied on a well characterized TRE, a direct repeat of AGGTCA separated by four nucleotides (DR4), using electrophoretic mobility shift assays and transient transfection in CV-1 cells. It was shown that RXR alpha blocked liganded TR alpha-induced transcription. However, ARP-1 did not block T3/TR alpha-mediated transcription, although both TR alpha/RXR alpha and TR alpha/ARP-1 heterodimers had similar affinities for the TRE-DR4. In contrast, wild type ARP-1 reversed the blocking effect of RXR alpha on the liganded TR alpha-induced transcription, whereas a dimerization domain-deleted ARP-1 mutant (ARP-1 delta C) did not, suggesting that ARP-1 modulates T3-induced transcription through a dimerization process. Thus, the TRE-DR4 differentially responds to T3 at the transcriptional level by accommodating a battery of TR heterodimeric complexes.

Thyroid hormone (T3) stimulates gene transcription by activation of thyroid hormone receptors (TRs), which bind to thyroid hormone response elements in T3-regulated genes. Retinoid-X receptors (RXRs), major members of the TR auxiliary proteins, have recently been shown to form TR-RXR heterodimers, to enhance the binding of the TRs to thyroid hormone response elements, and to augment TR-mediated transcriptional activation. In this report, we provide evidence that a putative adaptor(s), other than RXR, may be involved in T3-stimulated gene transcription. First, T3-stimulated, but not basal, transcription from the rat GH promoter was progressively and specifically inhibited by the addition of increasing amounts of either GST-TR beta or GST-RXR beta in a cell-free in vitro transcription. Second, this specific transcriptional inhibition is not due to disruption of the DNA-binding activity of the endogenous TR-RXR complex. These results suggest that inhibition of T3-stimulated transcription may be due to the sequestration of a limiting adaptor molecule(s). Hence, we hypothesize that a limiting adaptor(s), which may act as a bridging molecule between the TR-RXR complex and the basal transcriptional machinery, may be sequestered by either GST-TR beta or GST-RXR beta, resulting in the inhibition of T3-stimulated transcription.

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.

The GnRH receptor (GnRH-R) is a cell surface, G protein-coupled receptor that is highly expressed in pituitary gonadotropes. Activation of the receptor by GnRH stimulates the release of FSH and LH. Pituitary GnRH-R numbers and, hence, gonadotrope responsiveness to GnRH vary under different conditions and are regulated to a large extent by GnRH itself. To study the transcriptional regulation of the GnRH-R gene, a genomic clone containing 1.2 kilobases (kb) of the 5'-flanking region of mouse GnRH-R gene was isolated and characterized. A major transcriptional start site was identified 62 nucleotides upstream of the translational start site by primer extension and ribonuclease protection analyses. The promoter region does not contain canonical TATA sequences in the appropriate location. To determine whether this putative promoter is functional, it was subcloned into a luciferase reporter plasmid (GnRH-RLuc), and its transient expression was studied in cell lines of gonadotrope (alpha T3-1) and somatolactotrope (GH3) origins as well as those of nonpituitary origin (JEG-3 and CV-1). Luciferase activity was increased in alpha T3-1 (246-fold +/- 34.5-fold; P < 0.005) compared with the promoterless vector control but was considerably lower in GH3 (41-fold +/- 3.9-fold; P < 0.005), JEG-3 (12-fold +/- 0.9-fold; P < 0.005) and CV-1 (8-fold +/- 1.3-fold) indicating that GnRH-RLuc is preferentially expressed in cells of gonadotrope origin. Furthermore, GnRH agonist stimulated luciferase activity 3.4-fold +/- 0.3-fold (P < 0.005) above basal levels in GH3 cells cotransfected with rat GnRH-R complementary DNA, indicating that the GnRH-R promoter sequence is responsive to this ligand. In summary, we have identified and partially characterized the promoter region of the mouse GnRH-R and demonstrated that the regulatory elements for tissue-specific expression as well as for GnRH regulation are present within a 1.2-kb 5'-flanking region of the mouse GnRH-R gene.

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.

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.

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.

Follistatin, a glycosylated single chain protein that was originally isolated from ovarian follicular fluid, can specifically inhibit the biosynthesis and secretion of FSH by the pituitary. Follistatin has also been isolated from bovine pituitary and shown to have activin-binding activity. We wished to determine whether the follistatin gene is expressed in the rat pituitary and, if so, to identify the specific cell types. A 337-basepair fragment of the follistatin cDNA was amplified by polymerase chain reaction from a rat ovarian cDNA library and subcloned into pGEM3. Low levels of follistatin mRNA from rat pituitary poly(A)+RNA were detected by ribonuclease protection analysis using a specific follistatin riboprobe generated from the cDNA clone. The presence of follistatin mRNA in the pituitary was confirmed using polymerase chain reaction to amplify the follistatin cDNA generated by reverse transcription from total rat pituitary RNA. Furthermore, in situ hybridization studies combined with immunostaining for pituitary hormones were used to localize follistatin mRNA within the rat pituitary. When a biotinylated oligonucleotide complementary to follistatin mRNA was used with dispersed pituitary cells from rats in diestrus II, labeling was found in 5-7% of the cells. The in situ hybridization protocol was then combined with immunolabeling protocols for LH beta, FSH beta, or S-100 protein (a marker for folliculostellate cells). Follistatin mRNA was detected in 70 +/- 5% of LH beta cells, 44 +/- 11% of FSH beta cells, and 35 +/- 2% of folliculostellate cells. These results suggest that follistatin is expressed in pituitary gonadotropes and folliculostellate cells during diestrus II, where it may have a role in the local autocrine or paracrine regulation of FSH biosynthesis and secretion, possibly by binding to and modulating the effects of activin in the pituitary.

There are three known isoforms of the thyroid hormone receptor (TR) in the rat: TR alpha-1, TR beta-1, and TR beta-2. The TR alpha-1 and TR beta-1 mRNAs are found in many tissues, whereas TR beta-2 mRNA is detected only in the pituitary gland. Thus far, TR alpha-1 and TR beta-1 mRNAs have been found in humans and are highly homologous to their counterparts in rats; however, TR beta-2 mRNA has not yet been demonstrated in humans. To examine the expression of these TRs at the protein level, we have raised isoform-specific polyclonal antibodies in female New Zealand White rabbits against the rat TRs and c-erbA alpha-2, a carboxy-terminal variant of TR alpha-1 that does not bind thyroid hormone. The rabbits were immunized with synthetic peptides that contained the following amino acid sequences: TR alpha-common-(10-31), c-erbA alpha-2-(428-442), TR beta-1-(73-93), and TR beta-2-(86-101, 113-133). All immune sera could bind specifically to their respective immunizing peptides on enzyme-linked immunosorbent assay as well as immunoprecipitate specifically in vitro translated rat and human TRs. Anti-TR beta-1 and anti-TR alpha-common antibodies could immunoprecipitate TR beta-1 or TR alpha-1, respectively, in transfected COS-7 cells. We also immunostained normal adult rat and human pituitary glands. Each isoform-specific antibody could immunostain almost all of the anterior pituitary cells, suggesting that TR alpha-1, TR beta-1, TR beta-2, and c-erbA alpha-2 are most likely expressed in all anterior pituitary cell types in rats and humans. The staining of rat pituitary glands by the anti-TR beta-2 antibodies demonstrates for the first time that TR beta-2 is expressed as a protein in pituitary cells. Furthermore, the staining of human pituitary glands by the anti-TR beta-2 antibodies suggests that there is a human homolog of the rat pituitary-specific TR beta-2 that shares similar epitopes with the rat TR beta-2. In summary, we have prepared isoform-specific antibodies against TRs that can recognize in vitro translated, transiently transfected, and in situ rat and human pituitary TRs. These antibodies will be useful in examining tissue- and cell type-specific expression of rat and human TRs at the protein level.

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.

The effect of a single sc injection of the gonadal peptide, recombinant human activin A (rhActivin A), on gonadotropin synthesis and secretion was examined in adult and immature male and female rats and the effect of recombinant human inhibin A (rhInhibin A) was examined in adult male rats. Pituitary FSH beta, LH beta and alpha messenger RNA (mRNA) levels were determined by blot hybridization. Trunk blood was collected to measure serum FSH levels. Treatment with rhInhibin A (100 micrograms/kg) resulted in a decrease in FSH beta mRNA to 2% of controls levels 6 h after injection. FSH beta mRNA levels started to rebound at 10 h, but were still significantly lower than vehicle-treated controls. Serum FSH levels were significantly reduced at 2 h and were reduced further at 6 and 10 h. There were no significant changes in alpha and LH beta mRNA levels. RhActivin A, at the highest dose (500 micrograms/kg), in immature male rats had only a modest effect (1.2- and 1.3-fold increase) on FSH beta mRNA levels and FSH secretion, respectively, at 2 h. No increase in FSH synthesis and FSH secretion was observed in adult male rats. In contrast, both immature and adult-ovariectomized E2 implanted females showed a robust response to rhActivin A. In immature females, 2 h after rhActivin A (100 and 500 micrograms/kg) administration, FSH beta mRNA levels were elevated 2.0- and 2.2-fold. At this time serum FSH was also elevated. At 6 and 10 h rhActivin A significantly reduced FSH beta mRNA levels from vehicle-treated controls. In contrast, FSH secretion was elevated at 6 h and returned to baseline at 10 h. Administration of rhActivin A (500 micrograms/kg) to adult, ovariectomized-E2 females resulted in a significant increase in FSH beta mRNA levels and FSH secretion at 2 and 6 h. There were no significant changes in alpha and LH beta mRNA levels in either males or females. Thus, these in vivo studies have shown that rhInhibin A can inhibit FSH beta mRNA levels and FSH secretion in the adult male rat. RhActivin A stimulates FSH synthesis and secretion in the immature and adult ovariectomized-E2 females, but has little or no effect in immature and adult males. Hence, there is a sexual dimorphic response to rhActivin A in vivo in the rat.

Activin, a gonadal peptide, stimulates FSH secretion in association with an increase in FSH beta messenger RNA (mRNA) levels at the level of the anterior pituitary gland. The goal of these studies was to determine whether the effects of recombinant human activin A (rhActivin A) are exerted at the post-transcriptional level by affecting the stability of FSH beta mRNA. We determined the apparent half-life of FSH beta mRNA in the presence and absence of rhActivin A using actinomycin D. The anterior pituitary glands from adult male rats were isolated and dispersed enzymatically. Cells were preincubated in the presence of rhActivin A for 24 h to increase FSH beta mRNA levels. Actinomycin D was then added and the cells were incubated for a subsequent 4, 6, 8, 12, and 24 h in the presence or absence of rhActivin A. As reported earlier, the addition of rhActivin A caused parallel increases in FSH secretion and FSH beta mRNA levels, while having no effect on alpha or LH beta mRNA levels. Actinomycin D treatment decreased FSH beta mRNA to 49, 39, and 16% of control levels at the 4, 6, and 8 h time points, respectively. In contrast, when actinomycin D was added in the presence of rhActivin A FSH beta mRNA was reduced to 80, 58, and 42% of control levels at the 4, 6, and 8 h time points, respectively. Using the least squares method of analysis, the apparent half-lives of FSH beta mRNA under these two conditions were calculated. In the presence of actinomycin D, the half-life of FSH beta mRNA was 3.1 h. The addition of activin significantly increased the half-life to 6.5 h. These results suggest that activin A stimulates FSH beta mRNA levels, at least in part, at the posttranscriptional level by increasing the stability of FSH beta mRNA.

Galanin is a widely distributed regulatory peptide which modulates the pituitary secretion of PRL and GH. Estrogen administration strongly stimulates galanin gene expression in the rat anterior pituitary. In adult female Fischer 344 rats, estrogen also induces hyperplasia of lactotropes. We used immunocytochemical analysis to assess the effects of estrogen on galanin-like immunoreactivity (Gal-IR) in the rat pituitary and hypothalamus during sc diethylstilbestrol (DES) implantation and after its removal at 30 days. In the anterior pituitary, DES implantation increased the portion of Gal-IR-containing cells from less than 2% in the control rats to 18.3% after 3 days of DES and 36% after 30 days. These changes paralleled the lactotrope hyperplasia exhibited in response to DES exposure. Ten and 30 days after removal of the DES capsules, the percentage of Gal-IR-containing cells in the anterior pituitary decreased to 6.3% and 1.5%, respectively. Colocalization studies revealed that Gal-IR-containing cells were predominantly lactotropes. Immunoelectron microscopy demonstrated that Gal-IR was concentrated in the Golgi region of these hyperplastic lactotropes and suggests that little of the synthesized galanin is secreted. The distribution of Gal-IR in the hypothalamus, median eminence, and neurohypophysis was unaffected by DES treatment. These data demonstrate that galanin is synthesized by hyperplastic pituitary lactotropes of Fischer 344 rats and that peptide accumulation is dependent on the presence of circulating estrogens. In contrast, neuronal galanin synthesis in the hypothalamus does not appear to be regulated by estrogen.

Rat pituitary GH cells have been used extensively to study the biochemical actions of TRH on lactotropic cells. To investigate the structure and regulation of the rat TRH receptor (rTRHR), we have cloned its cDNA from GH4C1 cells. Using the polymerase chain reaction with degenerate primers and pools of cloned cDNAs from a GH4C1 cDNA library, a fragment sharing high similarity to the mouse thyrotrope TRHR (mTRHR) was identified. Conventional library screening with this fragment was used to isolate a single cDNA. mRNA synthesized in vitro from this cDNA was injected into Xenopus oocytes, and a characteristic conductance response to TRH was detected by voltage clamp recording. DNA sequence analysis revealed a molecule of 412 amino acid residues, with 96% similarity to the mTRHR. However, in contrast to the mTRHR, the rTRHR had an additional 19 amino acid residues at its carboxy-terminus. A mRNA of about 4 kilobases was identified in GH3 cells. Regulation of the rTRHR mRNA concentration was studied in GH3 cells. Steady state rTRHR mRNA levels were decreased to 30% of the control level by incubation with TRH for 48 h and increased 4-fold by incubation with dexamethasone for 12 h. Southern blot analysis of genomic DNA from GH3 cells gave a simple banding pattern consistent with a single copy gene. We conclude that the rTRHR shares high primary sequence similarity to the mTRHR, but the rTRHR has an extension of 19 amino acids at its carboxy-terminus, which is lacking in the mTRHR.

Using a sensitive method for the simultaneous measurement of TSH subunit mRNAs, we have investigated their hormonal regulation in the pituitary glands of normal and hypothyroid rats. Oligodeoxyribonucleotides (probes) complementary to coding regions of rat alpha- and TSH beta-subunit mRNAs were synthesized. These probes were 5'-end labeled with gamma-[32P] ATP and hybridized with total pituitary RNA obtained from T3-treated and untreated normal and hypothyroid rats. The samples were then exposed to S1 nuclease to digest single stranded nucleic acids. Specific hybridization of probes to the TSH subunit mRNAs would yield double stranded structures resistant to this enzyme. Measurement of the amount of undigested probes by denaturing polyacrylamide gel electrophoresis, autoradiography, and densitometry permits quantification of these mRNAs. Both rat alpha and TSH beta mRNAs were detected with as little as 0.1 microgram total pituitary RNA, representing a more than 10-fold increase in sensitivity compared to a standard RNA blot hybridization assay. Thyroidectomy resulted in a 3- to 5-fold increase, whereas T3 treatment caused a significant decrease in the subunit mRNAs in both normal and hypothyroid animals. However, in all treatment groups, the TSH beta mRNA was affected to a greater extent than the alpha mRNA by the changes in thyroid status. The ratio of alpha- to beta-subunit mRNAs was decreased with hypothyroidism and increased with T3 treatment. This assay allows simultaneous quantification of multiple mRNAs from a single pituitary gland within 48 h and should facilitate studies of the regulation of mRNAs encoding TSH subunits specifically and other pituitary proteins in general.

Adrenergic effects on TSH and subunit secretion were investigated in bovine anterior pituitary monolayer cultures. Epinephrine (E) (10(-6) M) caused a significant increase in TSH, alpha-subunit, and TSH beta release into the medium (P less than 0.001, P less than 0.001, and P less than 0.01, respectively). E, norepinephrine, and phenylephrine, all alpha-adrenergic agonists, caused significant increases in TSH release, with half-maximal effects at 4.3 X 10(-7), 6.8 X 10(-7), and 8.2 X 10(-7) M, respectively. However, isoproterenol, a beta-adrenergic agonist (10(-7)-10(-4) M), did not alter TSH or subunit release. Clonidine, an alpha 2-adrenergic agonist (10(-7)-10(-4) M), had no effect on TSH or subunit secretion; however, coincubation of clonidine (5 X 10(-7) M) with a submaximal concentration of phenylephrine (5 X 10(-7) M) caused a rise in TSH release greater (P less than 0.02) than that seen with P alone. The alpha-adrenergic antagonists phentolamine and fluphenazine completely inhibited (P less than 0.001) the E-induced rise in TSH and subunits. In contrast, the beta-adrenergic antagonists propranolol and metoprolol did not significantly inhibit the stimulation of TSH by E. TSH and subunit secretion is stimulated by adrenergic agonists acting directly on the pituitary, and this is probably mediated via an alpha-adrenergic receptor.

We hypothesized that if a CG similar in structure to other glycoprotein hormones was present in the rat placenta, then mRNAs encoding its subunits would be detectable. To investigate the possible presence of a CG in the rat, we attempted to detect mRNAs encoding the alpha- and CG beta-like subunits in the placentae of timed pregnant rats by sensitive blot hybridization analyses using cloned cDNAs encoding alpha- and beta-subunits of LH derived from rat pituitary mRNA. No subunit-specific mRNAs in placentae of pregnant rats at 4-21 days gestation were detected at either high or low stringencies of hybridization. However, under similar conditions, subunit-specific mRNAs were readily observed in total pituitary RNA of normal as well as ovariectomized rats. Moreover, hybridization with a cDNA for alpha-tubulin, a major component of the cytoskeleton, yielded easily detectable bands in rat placental RNA. In addition, hybridization analysis, under low stringency conditions, of restriction enzyme digests of rat genomic DNA with a rat LH beta-cDNA, which would detect LH beta subunit-like genes, suggests the presence of a single gene that, in fact, encodes the rat LH beta-subunit. We conclude that mRNAs encoding for proteins structurally homologous to rat LH subunits are absent in the rat placenta and that only a single LH beta-like gene is present in the rat. The luteotropic activity associated with the rat placenta must be related to a gonadotropin-like hormone whose structure is dissimilar from that of CG.

The effects of somatostatin (SRIF) on the production and release of TSH and its subunits have been investigated in bovine anterior pituitary monolayer cultures. SRIF caused a dose-dependent inhibition of TSH and subunit release by TRH, with a half-maximal effective dose of 3 X 10(-8) M. This effect was time dependent and was greater for TSH than for its subunits. However, the basal release and total production of TSH and its subunits over a 24-h period were not affected by SRIF. The effect of SRIF was additive to that of thyroid hormones in suppressing the release of TSH and its subunits by TRH. A combination of SRIF and thyroid hormone completely suppressed the release of TSH and its subunits by TRH. In contrast, thyroid hormones caused a dose-dependent inhibition of the total production, as well as the release, of TSH and its subunits induced by TRH. Furthermore, thyroid hormones produced a dose-dependent increase (r = 0.81; P less than 0.001) in the effectiveness of a single dose of SRIF in suppressing TSH release by TRH. Analysis of these data revealed that thyroid hormones interacted synergistically with the SRIF effect to suppress TRH-mediated TSH and subunit release.

We have previously demonstrated divergent changes in pituitary free TSH beta and alpha-subunit concentrations in hypothyroid mice during prolongation of experimental hypothyroidism and after T4 administration. This report compares the simultaneous responses of pituitary and thyrotropic tumor TSH, TSH beta, and alpha-subunit to 12 days of T4 administration in four groups of hypothyroid LAF1 mice bearing thyrotropic tumors. Half of each group received daily injections of T4 (10 micrograms/100 g BW, ip) for 12 days; the other half of each group received saline. Plasma concentrations of TSH, free TSH beta, and free alpha-subunit were suppressed by T4 administration to 0.1-1.5%, 2.0-3.9%, and 4.1-25% of control concentrations, respectively. Pituitary TSH and free TSH beta concentrations fell significantly with treatment to 24-43% and 10-28% of control concentrations, respectively. In contrast, pituitary alpha-subunit concentrations did not fall (they were 106-203% of control values), and a rise in the pituitary alpha-subunit concentration was statistically significant in one group (P less than 0.02). Thyrotropic tumor TSH and free TSH beta concentrations fell significantly with treatment to 9-31% and 8-35% of control concentrations, respectively. Tumor alpha-subunit concentrations did not fall (they were 87-195% of control values), and a 2-fold rise was statistically significant in one group (P less than 0.05). Both pituitary and tumor alpha-subunit to TSH beta molar ratios increased significantly with T4 administration in all groups. We conclude that (1) pituitary and thyrotropic tumor TSH beta concentrations fall after 12 days of T4 administration while alpha-subunit concentrations are unchanged or actually rise; (2) this divergent response is qualitatively similar in hypothyroid mouse pituitary and thyrotropic tumors; and (3) these data suggest differences between the regulation of intracellular TSH beta and alpha-subunit.

The modulation of gonadotropin gene expression in vivo by 17 beta-estradiol (E2) was investigated at the level of gonadotropin subunit gene transcription. Transcription rates were measured in isolated pituitary nuclei from untreated (control), ovariectomized (OVX), or estrogen-replaced (OVX-E2) female Sprague-Dawley rats. In control females, the average rate of alpha-subunit messenger RNA (mRNA) synthesis (161 +/- 30 ppm) was greater than the rates for either LH beta (42 +/- 17 ppm) or FSH beta (57 +/- 21 ppm). In OVX animals the synthesis rate for all three mRNAs was increased (alpha-subunit = 413 +/- 55 ppm; LH beta = 432 +/- 121 ppm; FSH beta = 204 +/- 91 ppm), but the 5- to 10-fold increases noted for LH beta were the most dramatic. Chronic E2 administration (10 micrograms/100 g BW sc daily for 7 days) suppressed these elevated transcription rates to near control values. The E2 effect occurred rapidly, with a significant suppression of all subunit transcription rates at 4 h, and a maximally suppressive effect at 24 h, at which time alpha-subunit = 30%, LH beta = 12%, and FSH beta = 26% of the mRNA synthesis rate in OVX animals. Thus, the estrogen status in vivo can affect gonadotropin subunit gene expression at the transcriptional level. Subunit gene transcription rates are regulated coordinately in a negative fashion, but LH beta mRNA synthesis is affected most dramatically.

Mouse pituitary thyrotropic tumors have been a useful model for the investigation of TSH and subunit regulation; however, little information has been obtained for the normal or hypothyroid mouse pituitary. Using antibodies against the purified mouse glycoproteins, we have developed RIAs for mouse TSH and its β-subunit which decreased or eliminated the nonparallelism routinely observed using rat assays. TSH, TSHβ, and α-subunit were measured in the plasma and pituitary of euthyroid mice and short term (41 days) and long term (13 months) hypothyroid mice with and without treatment with T4 (10 μg/100 g BW, ip) for 12 days. Plasma TSH, free TSHβ and free a concentrations increased progressively with prolongation of hypothyroidism, and treatment with T4 suppressed all three plasma concentrations in both hypothyroid groups. The pituitary TSHβ concentration rose 4-fold in short term hypothyroid mice (P < 0.001) and 23-fold in long term hypothyroid mice (P < 0.02). In contrast, the pituitary a concentration fell ...