Thyrotropin-releasing Hormone Receptor Messenger Research Articles

Expression of peptides and other neurochemical markers in hypothalamus and olfactory bulb of mice devoid of all known thyroid hormone receptors

We have investigated with histochemical techniques the expression of peptides and other neurochemical markers in the hypothalamus and olfactory bulb of male mice, in which the genes encoding the α and β thyroid hormone receptors (TRα1, TRβ1 and TRβ2) have been deleted. Thyrotropin-releasing hormone messenger RNA levels were increased in the hypothalamic paraventricular nucleus and in the medullary raphe nuclei of mutant mice lacking the thyroid hormone receptors α1 and β (α1 -/-β -/-), as compared to wild-type mice. In contrast, galanin messenger RNA levels were lower in the hypothalamic paraventricular nucleus of mutant animals, as was galanin-like immunoreactivity in the internal layer of the median eminence. Substance P messenger RNA levels were unchanged in the medullary raphe nuclei. Thyrotropin-releasing hormone receptor messenger RNA levels were increased in motoneurons, unchanged in the subiculum, and lower in the amygdala of mutant animals. Galanin messenger RNA levels were unchanged in the hypothalamic dorsomedial and arcuate nuclei of the thyroid hormone receptor α1 -/-β -/- mice, as was the immunocytochemistry for oxytocin and for vasopressin in the hypothalamic paraventricular nucleus. A reduction in tyrosine hydroxylase messenger RNA levels was found in the arcuate nucleus of mutant mice. In the olfactory bulb, immunohistochemistry for calbindin and for tyrosine hydroxylase revealed a reduction in the intensity of labeling of nerve processes in the glomerular layer of thyroid hormone receptor α1 -/-β -/- mice. The tyrosine hydroxylase messenger RNA levels were also slightly reduced. In contrast, the levels of galanin and neuropeptide Y messenger RNA in this region were unchanged in thyroid hormone receptor α1 -/-β -/- mice as compared to wild-type mice. Together these studies reveal many regional and neurochemically selective alterations in neuronal phenotype of mice devoid of all known thyroid hormone receptors.

Postnatal ontogeny of the thyrotropin-releasing hormone receptor messenger ribonucleic acids in the rat forebrain

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 level 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.

Estradiol transcriptionally and posttranscriptionally up-regulates thyrotropin-releasing hormone receptor messenger ribonucleic acid in rat pituitary cells

Estradiol transcriptionally and posttranscriptionally up-regulates thyrotropin-releasing hormone receptor messenger ribonucleic acid in rat pituitary cells

Estradiol transcriptionally and posttranscriptionally up-regulates thyrotropin-releasing hormone receptor messenger ribonucleic acid in rat pituitary cells.

17 beta-Estradiol (E2) has been shown to up-regulate the binding activity of the TRH receptor in rat pituitary cells. In this report, we investigated whether and how E2 alters TRH receptor expression at the mRNA level using the oocyte mRNA expression system, Northern blot analysis, and nuclear run-on assay. In oocytes injected with mRNA from the anterior pituitaries or GH3 cells, a TRH-induced 45Ca2+ efflux appeared. This efflux was dependent on the amount of TRH and injected RNA, and was inhibited by simultaneous addition of chloridazepoxide, an antagonist of the TRH receptor. Treatment of GH3 cells with E2 increased TRH receptor mRNA activity, as assessed in the oocyte expression system; the E2 effect became apparent after 3 h of treatment and reached a maximum (3- to 4-fold) between 6-72 h after addition. Northern blot analysis with a 412-basepair cDNA fragment or 3.7-kilobasepair full-length cDNA of the TRH receptor as a probe showed that E2 maximally (5-fold) increased the TRH receptor mRNA level of GH3 cells, with a half-maximal concentration of 0.1 nM after 6 h of treatment. The elevated level of mRNA induced by E2 was augmented, rather than impeded, by cycloheximide, indicating that ongoing protein synthesis was not required for the induction. The rate of transcription of the TRH receptor gene in isolated nuclei taken from GH3 cells was increased 3-fold by 2 h of treatment with E2. Furthermore, the half-life of the TRH receptor mRNA in GH3 cells was prolonged by E2 (from 2.3 to 4.4 h). These results demonstrate that E2 up-regulates the TRH receptors of the pituitary cells at the mRNA level by increasing both the transcription rate and stability.

Regulation of endogenous thyrotropin-releasing hormone receptor messenger RNA in GH4C1 cells: roles of protein and RNA synthesis

Regulation of endogenous thyrotropin-releasing hormone receptor messenger RNA in GH4C1 cells: roles of protein and RNA synthesis

Pituitary Adenomas of Patients with Acromegaly Express Thyrotropin-Releasing Hormone Receptor Messenger RNA: Cloning and Functional Expression of the Human Thyrotropin-Releasing Hormone Receptor Gene

We cloned a human thyrotropin-releasing hormone receptor (TRH-R) gene and its pituitary cDNA, and examined whether TRH-R mRNA may be expressed and function in the GH-secreting pituitary adenomas of patients with acromegaly. The human TRH-R consists of 398 amino acids and is a member of the G protein-coupled receptor family. The gene has a single intron in the coding sequence. An addition of TRH caused an obvious increase in intracellular calcium concentration in chinese hamster ovary cells expressing this receptor. Although highly homologous to the rat and mouse TRH-Rs, the human TRH-R has several differences in the carboxyl-terminal structure. The sequence and functioning analyses showed the same expression of TRH-R mRNA in the pituitary adenomas as that in the normal pituitaries, indicating that 1) some adenomas of patients with acromegaly express an intact and functional TRH-R, and that 2) the paradoxical response of GH secretion to TRH found in the patients might be a direct effect of TRH on TRH-R expressed in the adenoma.

Regulation of endogenous thyrotropin-releasing hormone receptor messenger RNA in GH4C1 cells: roles of protein and RNA synthesis.

In previous studies on the regulation of the endogenous TRH receptor (TRHR) in GH4C1 cells, we have shown that the synthetic glucocorticoid dexamethasone (Dex) increased TRHR abundance on the plasma membrane of these cells as well as increasing the concentration of TRHR mRNA. In the present investigation, we determined whether the action of Dex on TRHR mRNA was direct or whether it involved the synthesis of intermediary protein(s). We found that the protein synthesis inhibitors cycloheximide or anisomycin alone enhanced TRHR mRNA accumulation and, in the presence of Dex, caused a 10- to 20-fold superinduction of TRHR mRNA. The superinduction required inhibition of protein synthesis. Results of nuclear run-on assays showed that superinduction was accompanied by a large increase in transcription of TRHR mRNA. Experiments designed to examine the stability of TRHR mRNA transcripts revealed that inhibitors of RNA synthesis (actinomycin D and dichlorobenzimidazole riboside) caused an increase in endogenous TRHR mRNA concentrations by themselves, complicating their use to estimate TRHR mRNA half-life. We conclude that Dex stimulates transcription of endogenous TRHR mRNA by a mechanism that does not require enhanced synthesis of new proteins. In fact, ongoing protein synthesis reduces steady state TRHR mRNA concentrations consistent with a role for a labile protein(s) in suppressing TRHR gene transcription; an additional action of a labile protein to enhance TRHR mRNA degradation is also possible.

Transcriptional regulation by dexamethasone of endogenous thyrotropin-releasing hormone receptor messenger ribonucleic acid in rat pituitary GH4C1 cells.

Previous molecular studies on the mechanism of regulation of TRH receptor (TRHR) mRNA used transfected cells and concluded that the major mode of regulation is via mRNA degradation. Glucocorticoid hormones induce an increase in the number of TRHRs in rat pituitary GH cells, the model system used most extensively to characterize these receptors. We prepared a probe from the recently cloned rat lactotroph TRHR cDNA and investigated the mechanism of regulation by dexamethasone of endogenous TRHR mRNA in GH4C1 cells. Incubation with dexamethasone (100 nM for 6-48 h) induced a 3-to 4-fold increase in the steady state TRHR mRNA concentration (maximum at 24 h), as determined by Northern blot analysis. The stimulation was specific for glucocorticoids and dose dependent over the range 1-500 nM (maximum effect reached at 10 nM). The rate of transcription of the TRHR gene, measured by a nuclear run-on assay, was increased 3.0 +/- 0.57-fold (mean +/- SE) in cells treated for 12 h with 100 nM dexamethasone. We conclude that glucocorticoids regulate endogenous TRHR mRNA expression in GH4C1 cells in large part by controlling the rate of transcription of the TRHR gene.

Identification of thyrotropin-releasing hormone receptor messenger RNA in the rat central nervous system and eye

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.

The regional distribution of thyrotropin-releasing hormone receptor messenger ribonucleic acid in the brain

Messenger ribonucleic acid (mRNA) from mouse brain was reversely transcribed and complementary deoxyribonucleic acid (cDNA) encoding transmembrane domains (TM) 3 through TM6 of the thyrotropin-releasing hormone receptor (TRH-R) could be amplified by polymerase chain reaction (PCR). The partial cDNA of TRH-R was identical to that of the pituitary in size and sequence. The regional distribution of reverse transcription PCR products (partial cDNA of TRH-R) was similar to that of TRH-Rs themselves, except in cerebellum where TRH-R mRNA levels were relatively higher than the levels of TRH-Rs themselves previously reported.

Distribution of thyrotropin-releasing hormone receptor messenger RNA in the rat brain: An in situ hybridization study

Based on the recent cloning of the mouse thyrotropin-releasing hormone receptor, oligonucleotide probes complementary to the DNA sequence were constructed and used for in situ hybridization studies on the rat brain. Thyrotropin-releasing hormone receptor messenger RNA was found in many areas of the brain, mostly showing high degree of overlap with the distribution of thyrotropin-releasing hormone binding sites as previously revealed in autoradiographic studies. Thus, a strong signal was observed in the accessory olfactory bulb, the perirhinal sulcus, the ventral aspects of the hippocampal formation, some amygdaloid nuclei, the diagonal band nucleus, parts of nucleus accumbens, the bed nucleus of the stria terminalis, dorsomedial, lateral and perifornical hypothalamic regions, the septo-hippocampal nucleus, parts of the vestibular complex, as well as many bulbar motoneurons including the facial, dorsal vagal, ambiguus and hypoglossal nuclei, the superficial layer of the spinal trigeminal nucleus, and motoneurons and dorsal horn neurons in the spinal cord. Cells within one and the same nucleus expressed varying levels of thyrotropin releasing hormone receptor messenger RNA suggesting marked differences in rate of receptor synthesis. Most of these areas receive an input by thyrotropin-releasing hormone-positive nerve endings.Taken together these results suggest that thyrotropin-releasing hormone receptors are mostly localized in the vicinity of the cell bodies which express thyrotropin-releasing hormone receptor messenger RNA and mediate the wide range of actions that have been recorded after administration of exogenous thyrotropin-releasing hormone.

Mechanism of regulation of thyrotropin-releasing hormone receptor messenger ribonucleic acid in stably transfected rat pituitary cells

Mechanism of regulation of thyrotropin-releasing hormone receptor messenger ribonucleic acid in stably transfected rat pituitary cells

Mechanism of regulation of thyrotropin-releasing hormone receptor messenger ribonucleic acid in stably transfected rat pituitary cells.

We showed previously that the level of TRH receptor (TRH-R) mRNA in rat pituitary GH3 cells is down-regulated by TRH. Here, we study the mechanism of regulation of TRH-R mRNA in a line of GH3 cells that are stably transfected with mouse pituitary TRH-R cDNA (GH-mTRHR-1 cells). GH-mTRHR-1 cells were found to have 2.4 times the number of TRH-Rs and to stimulate a 2.5-fold greater increase in inositol phosphates in response to TRH than the parent cell line and to show TRH-induced down-regulation of TRH-R number. GH-mTRHR-1 cells contained 26 +/- 1.6 molecules of mouse TRH-R mRNA/cell and 230 +/- 31 molecules of mRNA for the neomycin resistance gene (NEO) with which it was cotransfected. In GH-mTRHR-1 cells, TRH caused a dose-dependent transient decrease in mouse TRH-R mRNA, with a nadir to 20% of control levels after 6 h. In contrast, TRH did not affect NEO mRNA or glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA, an endogenous gene product. TRH stimulated the rate of transcription of mouse TRH-R DNA by approximately 2-fold, but did not affect total poly(A) RNA synthesis. Most importantly, TRH caused a 4-fold increase in the rate of degradation of mouse TRH-R mRNA, but did not affect degradation of GAPDH mRNA. The half-lives of mouse TRH-R and GAPDH mRNAs were 3 and more than 20 h in control cells and 0.75 and more than 20 h in cells treated with 1 microM TRH for 1.5 h, respectively. These data show that the predominant effect of TRH on mouse TRH-R mRNA in GH-mTRHR-1 cells is to enhance the rate of its degradation. We suggest, therefore, that down-regulation of TRH-R mRNA caused by TRH in the parent GH3 cell line is secondary to increased TRH-R mRNA degradation.