The gene encoding human preproTRH was isolated from a human lung fibroblast genomic DNA library with a rat preproTRH cDNA fragment. The transcriptional unit is 3.3 kilobases in size and contains three exons interrupted by two introns of approximately 1050 and 650 base pairs, respectively. Exon 1 encodes the 5′untranslated region of the mRNA, exon 2 the putative signal sequence and the initial portion of propeptide, and exon 3 encodes the remainder of the propeptide, which contains six copies of the TRH sequence in contrast to five copies in the rat preproTRH gene. The predicted human preproTRH peptide structure has 242 amino acids compared to 255 amino acids in the rat. Homology with rat preproTRH is 73.3% and 59.5% at the nucleic acid and amino acid levels, respectively. Intron-exon splicing sites and 5′ and 3′ mRNA borders were confirmed rigorously by sequencing a human preproTRH cDNA using the polymerase chain reaction and human hypothalamic cDNA.

Graves' disease is the most common and most important cause of thyrotoxicosis. It occurs at any age and is approximately nine times more frequent in females than in males. The syndrome consists of one or more features: 1) thyrotoxicosis, 2) diffuse thyromegaly, 3) orbitopathy (exophthalmos), and rarely 4) dermopathy (pretibial myxedema and acropachy). There is a strong familial predisposition, and 15% of patients with Graves' disease have a close relative with the same disorder. The pathophysiology involves autoimmune mechanisms. T lymphocytes become sensitized to epitopes involving the TSH receptor of the thyroid gland. B lymphocytes are then activated to become IgG-producing plasma cells that generate thyroid-stimulating immunoglobulin (TSI) directed at the TSH receptor on thyroid cell plasma membranes. An analogous autoimmune process in the orbit leads to cell-mediated extraocular myositis and cellular infiltration of the retroorbital compartment, leading to exophthalmos, venous and/or lymphatic blockade, and secondary chemosis, injection, diplopia, and sometimes exposure keratitis which threatens vision. All of the ocular antigen(s) involved have not been elucidated, but a 64 kd protein, shared by the thyroid itself as well as by eye muscle, appears to be involved (1). Other epitopes may include the TSH receptor (TSH-R) itself, and TSH-R mRNA has been identified in orbital tissues.

Mechanisms of triiodothyronine (T3) negative regulation of the human thyrotropin-releasing hormone (TRH) gene were investigated with a chimeric construct of the 5' flanking region fused to a luciferase reporter gene, transfected into human neuroblastoma cells (HTB-11). Maximum negative regulation was achieved with constructs containing bases -242 to +54. Four sequences in this region exhibited homology with half sites of thyroid hormone response elements (TRE) (AGGTCA). The most important site was a sequence with an overlapping TRE/CRE, involving bases -53 to -60 (TGACCTCA). Potential combinatorial interactions of thyroid hormone receptors and CREB at this site were explored. Modest promoter stimulation was achieved with dibutyryl cyclic adenosine monophosphate (cAMP) (10(-3) M) plus IBMX (0.5 mM). Stimulation was greatly enhanced (+820%) by cotransfection of a constitutively activated protein kinase A (pPKA) construct. Cotransfection with pCREB increased stimulation further to 1350% above control. Stimulation of pPKA and pCREB interfered with stimulation by unliganded TRbeta1, and co-transfected pPKA and pCREB blocked T3 negative inhibition by TRbeta1-T3 complexes. When this site was mutated by polymerase chain reaction (PCR) mutagenesis, the mutant construct failed to respond to unliganded TRbeta1, and stimulation by pPKA and/or pCREB was inhibited markedly, from 12.5- to 2.1-fold, p < 0.001. Moreover, TRbeta1-T3 complexes failed to show any inhibition of the mutated promoter. These results suggest that negative regulation is achieved by inhibition of CREB stimulation of the TRH promoter at this overlapping TRE/CRE site. The two cosuppressors, NCoR and SMRT, were able to augment stimulation of the TRH promoter by unliganded TRbeta1 and enhance the magnitude of T3 inhibition. The potential role of the TRH gene and the pathophysiology of thyroid hormone resistance was investigated with three mutant TRbeta1 constructs. Thyroid hormone resistance was found to be expressed at the level of TRH gene regulation, due to lowered inhibition by mutant TRbeta1-T3 complexes and by their dominant negative effects on wild-type TRbeta1-T3 inhibition. TRH gene expression has been identified in the heart. Cardiac TRH mRNA was not regulated by T3, in contrast to HTB-11 cells, but cardiac TRH mRNA density could be augmented by glucocorticoids and by testosterone. TRH receptors were identified using Scatchard blots that showed a kilodalton of 1.4 nM and a bmax of 10 pmol/mg protein. TRH-R mRNA was identified also by reverse transcription polymerase chain reaction (RT-PCR). Enhanced ventricular contractility by TRH was demonstrated in both an open-chested dog preparation and in ex vivo ventricular myocytes, using video edge cinematography. Under controlled conditions, myocyte shortening was 13.3%, and TRH (10(-6) M) caused muscle shortening to increase 140%, (p < 0.005). TRH gene expression was demonstrated exclusively in Leydig cells of the testis. High affinity binding sites were identified in testicular membranes with a kilodalton of 1.6 x 10(-6) M. TRH was able to inhibit LH and HCG-activated testosterone secretion significantly. Thus, one paracrine role of TRH in the testis may be to serve as inhibitory modulator of gonadotropin-stimulated testosterone secretion.

Postnatal developmental change of the thyrotropin-releasing hormone receptors (TRHR) in the rat forebrain was investigated using TRH binding assays and Northern blot analyses from postnatal day 8 to the age of 2 years. TRH binding assays, with [3H]MeHisTRH as the radioactive ligand, demonstrated that the binding capacity in the forebrain was lowest at postnatal day 8 and increased to a maximum level at postnatal day 20. The TRH binding significantly decreased to adult levels between days 20 and 35, and no significant change was observed thereafter. Northern blot analysis, with a 32P-labeled TRHR cRNA probe, revealed that expression of the TRHR gene in the forebrain was not detectable on day 8 after birth, whereas apparent gene expression could be detected in the anterior pituitary. In contrast to the binding capacities, TRHR mRNA levels were very low until postnatal day 20, and increased significantly between days 20 and 35. No significant alteration in mRNA levels was observed after day 35. These results indicated that: (1) TRH binding capacities in the forebrain increased to a maximum levels between the second and third postnatal week and thereafter decreased to adult level, (2) the levels of TRHR mRNA and the TRH-binding capacities did not correlate in that period, suggesting that the TRHR number in the immature forebrain might be regulated by a posttranscriptional mechanism, and (3) expression of the TRHR gene in the forebrain and pituitary seemed to be regulated differentially during development.

TRH is negatively regulated by T3 both in the hypothalamic paraventricular nucleus and transient transfection models. Mutations in hTR beta 1 genes are associated with the syndrome of generalized resistance to thyroid hormone. To investigate potential effects of mutant TRs on T3 regulation of the hTRH gene, transient gene expression assays were performed in human neuroblastoma (HTB-11) cells with an hTRH promoter-luciferase construct, wild type (WT) hTR beta 1, and three qualitatively distinct hTR beta 1 mutant forms (ED, OK and PV). In the presence of T3 (10(-9) M), liganded WT-hTR beta 1 inhibited hTRH promoter activity significantly (40%). Cotransfection of each of the two mutants (ED and OK) achieved similar levels of inhibition only at 10 to 100 fold increased T3 concentrations. Of interest, a 10x excess of mutant ED or OK could also exert dominant negative effects upon WT hTR beta 1-T3 mediated inhibitory actions on the hTRH promoter. In contrast, mutant TR-PV exerted neither inhibitory nor dominant negative effects at even higher concentrations of T3. Moreover, all three unliganded mutant forms stimulated TRH promoter activity significantly in the absence of T3, despite their different mutations in the ligand-binding domain (LBD). These data demonstrate that thyroid hormone resistance at the level of TRH gene regulation, due to reduced inhibitory actions of mutant TR-T3 complexes, as well as dominant negative effects upon WT hTR beta 1 mediated inhibition, likely contribute to elevated TSH values observed in the syndrome of thyroid hormone resistance.

Thyrotropin-releasing hormone (TRH) is the key regulator of the synthesis and secretion of TSH in animals and humans (Wilber and Yamada 1990). The biological implications of this peptide, the first releasing hormone to be characterized, has generated a large literature regarding both pituitary TSH and extrapituitary roles of TRH as a neurotransmitter and/or neuro-modulator in the central nervous system (O'Leary and O'Connor 1995, Morley 1981). In this review, new areas of TRH biology are explored, focused on the differential regulation of the TRH gene by triiodothyronine (T3) and other substances in the hypothalamus and two unexpected extrahypothalamic loci, the heart and testis. These new directions should enlarge our understanding concerning how hormones like T3 regulate genes negatively and selectively with the identical receptors and DNA elements required for positive gene stimulation. In addition, regulatory studies of the TRH gene by T3 should be relevant to other hormone receptor interactions with DNA sequences in general, as glucocorticoids, mineral-ocorticoids, sex steroids, vitamin D, and retinoic acid are ligands for homologous receptor proteins in the nuclear receptor superfamily.

The thyrotropin-releasing hormone (TRH) gene is regulated negatively at the transcriptional level by thyroid hormone (T3). T3 positive regulatory effects on other target genes, such as the growth hormone gene, are mediated through heterodimerization of thyroid hormone receptors (TRs) with RXR or other auxiliary nuclear protein(s). To explore whether an accessory co-suppressor protein(s) may be involved in T3 inhibitory regulation of human TRH gene transcription, transient gene expression studies have been carried out using a hTRH-luciferase (TRH-Luc) chimetric reporter construct, an hTR beta 1 expression construct, and pABgal-hTR beta 1 ligand-binding domain (LBD) fusion constructs, cotransfected into a human neuroblastoma cell line (HTB-11,ATCC). Results herein indicate that T3-dependent inhibitory regulation (48-60% of control) of the hTRH gene promoter by hTR beta 1-T3 complexes could be abrogated completely by cotransfection of a 10 x excess of hTR beta 1-LBD (TR 168-456 aa) in a pABgal94 vector. In striking contrast, cotransfection of a 10 x excess of highly truncated hTR beta 1-LBD (TR 452-456 aa) failed to reverse T3-mediated TRH promoter inhibition. This squelching effect by excessive intact TR-LBD, moreover, could not be reversed by raising T3 concentration 100-fold (from 10(-8) to 10(-6) M), thus excluding a squelching effect of T3 itself by excess LBD. These results suggest that negative regulation of the hTRH gene promoter activity by TR beta 1-T3 complexes involves interactions with an accessory co-suppressor protein, which may bridge DNA-bound TR beta 1-T3 complexes to the transcriptional initiation complex.

Thyroid hormone (T3) and estradiol (Est) modulate biological processes by binding to nuclear receptor proteins that, through interactions with specific response elements in the regulatory regions of genes, modulate gene transcription. Est stimulation of estrogen receptor (ER)-positive breast carcinoma cell growth occurs through its ability to bind to the ER and activate gene transcription. We now report that physiological concentrations of T3 significantly enhance Est stimulation of growth of a number of human breast carcinoma cell lines. The effect of T3 is specific for Est stimulation of growth and has no effect on insulin-like growth factor-I stimulation of growth. The effect of T3 on enhancing Est-mediated growth was specifically blocked by the addition of ligands inducing retinoid X receptor (RXR) homodimer receptor formation, suggesting that RXR-thyroid nuclear receptor (TR) heterodimer formation is required for the T3-mediated effect on estradiol-stimulated growth. Four thyroid nuclear receptors have been described in tissues, TR alpha 1, alpha 2, beta 1, and beta 2. Breast carcinoma cells were found to express TR beta 1 and TR alpha 2 mRNA and very low levels of TR alpha 1 mRNA. T3 did not increase ER mRNA or protein levels and did not enhance Est-mediated increases in gene transcription of a number of genes, i.e., transforming growth factor-alpha and pS2 which contain estrogen-response elements (EREs) in their regulatory regions. However, T3 enhanced Est-stimulated ERE-TK-CAT activity. Thus significant cross-talk appears to occur between the TRs and ER and T3 appears to enhance Est-mediated gene transcription.

We have recently documented the expression of preprothyrotropin-releasing hormone (TRH) gene in murine, human and rat testis. Moreover, we have localized TRH to rat Leydig cells immunohistochemically, and found that both prepro TRH mRNA and TRH levels are developmentally regulated in the rat testis. To investigate the potential roles of TRH in testicular function, characterization of TRH receptors (TRH-R) in this tissue was undertaken. Recently, a cDNA encoding murine TRH-R has been isolated, making possible cloning of a rat TRH-R cDNA from the anterior pituitary gland. This cDNA was used for detection of TRH-R gene expression in the rat testis by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). TRH receptor assays were also performed with (3H)MeHisTRH as the radioactive ligand. In Northern blot analysis, a single and specific hybridization band, approximately 3.8 kb in size, was identified in whole testis RNA, identical in size with that found in the anterior pituitary gland. The concentration of TRH-R mRNA in the testis was approximately 10% of that in the pituitary. TRH-R mRNA was also detected by RT-PCR in Metrizamide gradient-purified Leydig cells. TRH receptor binding assays revealed the presence of specific, high affinity binding sites with a Kd of 1.6 x 10(-8) M in the testis. Such TRH binding was inhibited by chlordiazepoxide, a specific antagonist of TRH receptor binding. We conclude that TRH may exert local, probably autocrine, actions in the testis via a transmembrane receptor very similar or identical to that in pituitary.

Thyrotropin releasing hormone (TRH) gene is regulated negatively at the transcriptional level by thyroid hormone (T3) in rat anterior hypothalamus. The actions of T3 upon other target genes are known to be mediated through the thyroid hormone receptors (TR), TRα and TRβ. To explore whether the inhibitory regulation of human (h) TRH gene transcription by T3 is TR isoform specific and whether TRH gene transcription can be modulated as well by unliganded TR isoforms, transient gene expression studies have been carried out using hTRH-luciferase (TRH-Luc) chimeric constructs and TR expression constructs, co-transfected into a human neuroblastoma cell line (HTB-11). Data herein demonstrate T3-dependent inhibitory regulation of the hTRH gene promoter by TR-T3 complexes. Moreover, significant inhibition (39%-60%) could be achieved by T3 bound to either hTRα1, hTRβ1, or rTRβ1, β2 and was comparable quantitatively, indicating an absence of TR isoform specificity for T3 inhibition. Conversely, basal promoter activity of the hTRH gene could be activated significantly by unliganded hTRα1, β1, rTRβ1, and β2 (150% to 334%), but not by hTRα2. Thus, TRs appear to exert opposite effects on hTRH gene transcription, depending on the presence or absence of ligand T3. These dual effects of TR suggest that the addition of the T3 ligand effects conformational changes that can abrogate the initiation of transcription.

Using the reverse transcription-polymerase chain reaction (RT-PCR), a cDNA encoding the entire rat thyrotropin-releasing hormone receptor (TRH-R) was isolated from normal rat pituitary gland mRNA. In addition, a novel truncated isoform of TRH-R which lacks 52 base pairs (bp) in the carboxyl (C-) terminal tail was isolated. This truncation, probably generated by alternate splicing, causes a frame-shift and results in a truncated TRH-R 25 amino acids shorter and with a different C-terminal amino acid sequence than the longer type receptor. This truncated TRH-R mRNA, along with the longer receptor form, was found to be expressed throughout the rat pituitary gland and brain.

TRH exerts a wide variety of neuropharmacological actions by interacting with specific receptors in the central nervous system (CNS). Specific binding sites for TRH have been identified also in the mammalian retina. However, whether TRH receptors (TRH-R) in the brain and retina are identical in structure with those in the anterior pituitary gland is presently unknown. In this study, TRH-R gene expression was examined by Northern blot analysis in the CNS and eye using a cloned rat pituitary TRH-R cDNA. Northern analysis demonstrated a specific hybridization band of approximately 3.8 kb in hypothalamus, cerebrum, cerebellum, brain stem, spinal cord, and eye, indistinguishable from that characterized in pituitary gland. These data strongly support the hypothesis that a TRH receptor similar or identical to that cloned from the pituitary occurs in the retina and throughout the CNS.

Thyrotropin-releasing hormone (TRH) plays the central regulatory role in the hypothalamic-pituitary-thyroid axis, but is also present in many extra-hypothalamic loci. The adult rat testis has been identified previously as a source of hypothalamic neuropeptides including TRH. To investigate whether the TRH gene is transcribed in testis, the identification and localization of prepro(pp) TRH mRNA and TRH were studied. Northern blot analyses of ppTRH mRNA in the adult rat testis showed a 2.0 kb band, hybridized with a ppTRH cRNA probe. This band was 0.4 kb greater than the 1.6 kb hypothalamic band. The concentration of ppTRH mRNA in the adult testis was approximately 13% of that found in the hypothalamus. Developmental studies of testicular ppTRH mRNA revealed that no ppTRH mRNA could be detected at the earliest stage (day 8). However, hybridization signals were detected on day 20 and increased progressively on days 35, 45 and 70 by 5.8, 6.4, and 9.8-fold, respectively. In addition, ppTRH mRNA was determined in Leydig cells by Northern analyses of elutriated testicular cell fractions. TRH was also measured in the rat testes at different developmental stages by RIA. TRH concentrations paralleled ppTRH mRNA during development. TRH was localized to Leydig cells by immunohistochemistry. These results indicate that ppTRH mRNA and TRH are present in the rat testis, especially in the Leydig cells. The changes of ppTRH gene expression and the concentration of TRH in the rat testis are developmentally dependent. TRH may function as a new paracrine or autocrine regulator of testicular function.

To gain further insight into the regulation of hypothalamic TRH by thyroid hormones, we measured TRH concentration in specific hypothalamic nuclei and preproTRH mRNA levels in the anterior hypothalamus. Adult male rats were decapitated 1, 7, 14 days after thyroidectomy. Micropunches by the method of Palkovitz from seven hypothalamic nuclei and median eminence were used for measurement of TRH by radioimmunoassay. As compared with normal levels, TRH concentration significantly decreased in the median eminence and five hypothalamic nuclei including paraventricular nucleus (PVN), posterior nucleus, anterior nucleus, arcuate nucleus, and ventromedial nucleus pars lateralis, by 7 days after thyroidectomy. No significant changes were observed in dorsomedial nucleus or ventromedial nucleus pars medialis until 14 days after thyroidectomy. A rapid and simple method to detect specific mRNA for preproTRH was developed using the polymerase chain reaction and a single anterior hypothalamic section. PreproTRH mRNA levels in the anterior hypothalamus increased approximately twice 7 days after thyroidectomy. These data indicate that thyroidectomy caused a marked increase in preproTRH mRNA levels of the anterior hypothalamus, while it significantly reduced TRH concentrations not only in PVN and median eminence but also in other specific hypothalamic nuclei, suggesting that these nuclei might be involved in the thyrotropin regulation in the hypothalamus.

Our laboratory has demonstrated recently that thyrotropin (TSH) secretion is regulated in part by thyroid hormone inhibition of TSH-releasing hormone (TRH) synthesis and secretion, both in vitro and in vivo. Minute amounts of triiodothyronine (T3) can suppress TSH secretion when administered intracerebraventricularly, whereas identical quantities of T3 administered by a peripheral route do not lower circu-lating TSH concentrations (1). In addition, TRH secretion is augmented in vitro from experimentally hypothyroid rats and secretion is below normal from thyrotoxic rat hypothalami, and T3 (10-9-10-1 M) intro-duced into incubational media can prevent ouabain-activated TRH secretion in vitro (2). Most recently, we have demonstrated that there is augmented TRH messenger RNA in experimentally hypothyroid rats and reduced messenger RNA in the hypothalamic paraventricular nucleus in thyrotoxic rats, whereas no changes were seen in either group when whole hypothalamic RNA was hybridized (1). On the basis of these considerations, we wanted to determine whether thyroid hormones were involved also in the inhibitory regulation of the human TRH gene. Two X 106 plaques of a human lung fibroblast Lambda Fix genomic library were screened with a 32P-cDNA rat preproTRH (ppTRH) gene probe, which included the last three TRH coding sequences of the third exon. Under conditions of moderate stringency, an initial 15 kb DNA fragment was identified, which included the complete ppTRH genomic sequence of 3.3 kb, containing three exons separated by two introns of 1,050 and 650 base pairs respectively Figure 1 (3). The inferred cDNA sequence was confirmed by PCR amplification of hypothalamic

PreproTRH mRNA has been identified in rat cardiac tissues by Northern analyses and RNase protection assays with a specific rat 32P-TRH cRNA probe. Densitometric analyses revealed that atrial ppTRH mRNA concentrations were approximately five-fold greater than those of the ventricles. TRH concentrations (RIA), by contrast, were two-fold higher in ventricles. These data suggest that TRH and TRH mRNA are present in the rat heart, but their concentrations are dissociated, possibly because of differential post-transcriptional or post-translational processing. TRH is postulated to play an autocrine or paracrine role in cardiac physiology.

Changes in circulating cyclo(His-Pro) (CHP) levels after ingestion of 0.5 g/kg of glucose (oral) were measured in 32 Sprague Dawley rats at varying time points between 0 (before glucose) and 60 minutes. Each rat provided one time point. CHP levels displayed a biphasic response rising from 933 +/- 56 pg/ml to 1083 +/- 287 pg/ml (p greater than 0.05), followed by a fall to 543 +/- 46 pg/ml (p = 0.002) and then a recovery to near baseline values. To further investigate this response, a separate group of rats were sampled sequentially after 3 g/kg oral glucose (n = 7), 0.6 g/kg I.V. glucose (n = 6) or controls given saline only (n = 14). CHP levels were significantly elevated in the oral glucose group at 5 (p = 0.048) and 12 (p = 0.02) minutes compared to controls, while the i.v. glucose group was not statistically different from controls. Comparison of the mean of the highest incremental response to baseline values in each group revealed a greater excursion in CHP levels after oral (p = 0.027) glucose than after I.V. (p = 0.08) glucose. These data suggest CHP is acutely and reversibly elevated after glucose ingestion in rats and that the response is greater after oral glucose than after i.v. glucose.