Regulation of thyrotropin-releasing hormone receptors is cell type specific: comparison of endogenous pituitary receptors and receptors transfected into non-pituitary cells.

TRH, which does not elevate cyclic AMP, and elevation of cellular cyclic AMP decrease the density (down-regulate) of TRH receptors (TRH-Rs) on pituitary (GH3) cells. In this study we measured the effects of TRH and elevation of cyclic AMP on TRH-Rs expressed in non-pituitary cells transfected with a recently cloned mouse pituitary TRH-R complementary DNA. In stably transfected rat glioma (C6-2) cells and transiently transfected COS-1 cells TRH caused TRH-R down-regulation while elevation of cyclic AMP caused increases in TRH-R density. Hence, the effects of cyclic AMP on TRH-Rs in transfected C6-2 and COS-1 cells are different from those in GH3 cells while the effects of TRH on TRH-R are similar in all three cell types. These data show that regulation of TRH-Rs is cell type specific.     

Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor.

Thyrotropin-releasing hormone (TRH) is an important extracellular regulatory molecule that functions as a releasing factor in the anterior pituitary gland and as a neurotransmitter/neuromodulator in the central and peripheral nervous systems. Binding sites for TRH are present in these tissues, but the TRH receptor (TRH-R) has not been purified from any source. Using Xenopus laevis oocytes in an expression cloning strategy, we have isolated a cDNA clone that encodes the mouse pituitary TRH-R. This conclusion is based on the following evidence. Injection of sense RNA transcribed in vitro from this cDNA into Xenopus oocytes leads to expression of cell-surface receptors that bind TRH and the competitive antagonist chlordiazepoxide with appropriate affinities and that elicit electrophysiological responses to TRH with the appropriate concentration dependency. Antisense RNA inhibits the TRH response in Xenopus oocytes injected with RNA isolated from normal rat anterior pituitary glands. Finally, transfection of COS-1 cells with this cDNA leads to expression of receptors that bind TRH and chlordiazepoxide with appropriate affinities and that transduce TRH stimulation of inositol phosphate formation. The 3.8-kilobase mouse TRH-R cDNA encodes a protein of 393 amino acids that shows similarities to other guanine nucleotide-binding regulatory protein-coupled receptors.     

Evidence for dual regulation by protein kinases A and C of thyrotropin-releasing hormone receptor mRNA in GH3 cells.

We showed previously that TRH down-regulates TRH receptor (TRH-R) mRNA in GH3 cells by a mechanism that appears to be mediated by protein kinase C. Here we show that vasoactive intestinal peptide (VIP) down-regulates TRH-R mRNA and present evidence that this action is mediated by protein kinase A. In GH3 cells, VIP caused a time- and concentration-dependent decrease in TRH-R mRNA. This VIP effect was simulated by 8-(4-chlorophenylthio)-cAMP, forskolin, cholera toxin and 1-methyl-3-isobutylxanthine. When cells were incubated with agents that elevate cAMP and TRH or phorbol 12-myristate 13-acetate, the decrease in TRH-R mRNA was greater than with either agent alone. When cells were pre-incubated with H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, the effects of VIP, TRH and phorbol 12-myristate 13-acetate were inhibited. We suggest that VIP, via protein kinase A, and TRH, via protein kinase C, dually regulate TRH-R mRNA.     

Thyrotropin-releasing hormone stimulation of phosphoinositide hydrolysis desensitizes. Evidence against mediation by protein kinase C or calcium.

Previous reports have provided conflicting evidence as to whether the response to TRH desensitizes. Here we show that TRH stimulation of phosphoinositide (PPI) hydrolysis, measured as inositol phosphate accumulation in the presence of LiCl, desensitizes in rat pituitary GH3 cells and in rat glioma C6 cells stably transfected with mouse pituitary TRH receptor complementary DNA. In GH3 cells, the rate of stimulation by 1000 nM TRH of PPI hydrolysis was maximal initially and then decreased by 44 +/- 13% after 20 min. In an experimental paradigm in which PPI hydrolysis was measured by adding 20 mM LiCl at different times after TRH, desensitizations caused by 3, 10, and 1000 nM TRH were 33 +/- 5%, 41 +/- 6%, and 69 +/- 2%, respectively. In transfected C6 cells, TRH-induced desensitization of 76 +/- 9% was found. In GH3 cells, 1 microM phorbol myristate acetate (PMA), an activator of protein kinase C, inhibited the initial response to TRH by 75 +/- 6% and preexposure to PMA and TRH decreased the rate of PPI hydrolysis by 98 +/- 1% after 60 min. One hundred micromolar H-7 (1-(5-isoquinolinesulfonyl)-2-methyl piperazine), an inhibitor of protein kinases, abolished the effect of PMA but did not inhibit TRH-induced desensitization. Elevation of cytoplasmic free Ca2+ by K+ depolarization increased TRH stimulation of PPI hydrolysis. We conclude that TRH stimulation of PPI hydrolysis acutely desensitizes and that this effect is not specific to pituitary cells. TRH-induced desensitization, moreover, does not appear to be mediated by protein kinase C or by elevation of cytoplasmic free Ca2+.     

Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin-releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells.

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.     

Stimulatory effect of pituitary adenylate-cyclase activating polypeptide (PACAP) and its PACAP type I receptor (PAC1R) on prolactin synthesis in rat pituitary somatolactotroph GH3 cells

In this present study, we investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor, PACAP type I receptor (PAC1R) on prolactin synthesis in pituitary somatolactotroph GH3 cells. PACAP increased prolactin promoter activity up to 1.3 ± 0.1-fold. This increase, while significant, was less than the increase resulting from thyrotropin-releasing hormone (TRH) stimulation. By transfection of a PAC1R expression vector to the cells, the response to PACAP on prolactin promoter activity was dramatically potentiated to a degree proportional to the amount of PAC1R transfected. In the PAC1R expressing GH3 cells, TRH and PACAP alone increased prolactin promoter up to 3.3 ± 0.3-fold and 4.9 ± 0.2-fold, respectively, and combined treatment with TRH and PACAP further increased prolactin promoters up to 6.8 ± 0.6-fold. PACAP binds both Gs- and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/extracellular signal-regulated kinase (ERK) signaling pathways. PACAP increased ERK phosphorylation in PAC1R expressing cells to the same degree as TRH. Combined treatment with TRH and PACAP had a synergistic effect on ERK activation. GH3 cells produce both prolactin and growth hormone. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin and attenuated growth hormone mRNA expression. PACAP increased both prolactin and growth hormone mRNA levels, particularly in PAC1R expressing cells. In addition, increasing amount of PAC1R in GH3 cells potentiated the action of TRH on prolactin promoter activity, as well as on ERK phosphorylation. PAC1R was induced by PACAP itself, but not by TRH. Our current study demonstrates that PACAP and its PAC1R, functions as a stimulator of prolactin alone or with TRH in prolactin producing cells.     

TRH is a tonic secretagogue in growth hormone secreting but not in nonfunctioning pituitary tumors

In most patients with growth hormone (GH) secreting pituitary adenomas and clinically nonfunctioning pituitary tumors (NFPT) the intravenous injection of thyrotropin releasing hormone (TRH) augments the secretion of GH and subunits of gonadotropin hormones respectively. Similar hormone responses to TRH have been detected in rat pituitary cell lines and in primary human pituitary tumor cultures in vitro. Nevertheless the TRH effect on tumor hormonal secretion has not been well characterized. In the present study we examined TRH-induced hormone secretion in GH secreting tumors and in NFPT in vitro. Cultured cells secreted betaLH and betaFSH (NFPT) or GH (GH secreting adenomas) up to 14 days in culture. In NFPT TRH (10(-8) mol/l) elicited peak betaLH and betaFSH secretion at 60 to 90 min, with no further increase at 24 h. TRH-stimulated GH secretion peaked at 90-120 min, and decreased after 3 h, but a secondary rise occurred after 24 h of incubation. Chronic daily exposure to TRH followed by an acute TRH challenge resulted in a further increase of GH secretion after one hour. In contrast, acute TRH administration following chronic exposure did not elicit increased P-subunits secretion in NFPT. Coadministration of cycloheximide did not change TRH induced beta-subunits secretion in NFPT. However, when it was administered 24 h prior to TRH, it blocked both basal and TRH induced beta-subunits levels in NFPT. Cycloheximide had no effect on basal or stimulated GH secretion when administered concomitantly or 24 h before TRH. Incubation of cultured GH secreting tumors with cycloheximide during 5 days blocked both basal and TRH stimulated GH secretion, thus indicating dependency on protein synthesis during the chronic, secondary phase. Since the acute secretion was not affected by coadministration of cycloheximide, these early increases in hormone levels apparently reflect the release of stored hormone. In summary, GH secreting adenomas and NFPT differ significantly in their hormonal response to continuous exposure to TRH. The mechanisms underlying the sustained effect of TRH on GH secretion in vitro remain to be investigated. If endogenous TRH exerts a similar continuous effect it may contribute to the disregulated GH secretion in acromegaly.     

Autoregulation of pituitary growth hormone messenger ribonucleic acid levels in rats bearing transplantable mammosomatotrophic pituitary tumors

Female Wistar-Furth rats were implanted sc with GH3 rat pituitary tumor cells. Tumors were palpable by 4 weeks, and animals were killed periodically from 5-9 weeks. Tumor-bearing rats (n = 10) were heavier than their respective controls, reaching a weight of 372 +/- 3 by 9 weeks vs. 195 +/- 5 g in controls (mean +/- SE). Circulating serum GH levels increased in tumor-bearing animals from 218 +/- 50 to 9067 +/- 962 ng/ml. Serum insulin-like growth factor I (IGF-I) levels were elevated 3-fold in tumor-bearing rats. After death, pituitary glands were excised, and their total RNA was extracted. GH mRNA was assayed by dot hybridization of immobilized pituitary RNA with [32P]cDNA for rat GH. The hybridization signal was quantified by densitometry of autoradiographs. Pituitary rat GH mRNA levels were suppressed 50% in tumor-bearing animals after 5 weeks. By the end of the 9-week period, pituitary GH mRNA levels were undetectable in tumor-bearing animals. The results show that GH tumor-bearing animals exhibit high levels of circulating GH and IGF-I and suppressed endogenous pituitary GH mRNA levels. This may be caused by autoregulation of pituitary GH gene expression either at the level of the hypothalamus or by a direct effect of GH on the pituitary. Alternatively, the elevated levels of IGF-I may be responsible for the suppression of pituitary GH gene expression .     

Epidermal growth factor decreases the concentration of thyrotropin-releasing hormone (TRH) receptors and TRH responses in pituitary GH4C1 cells.

Treatment of pituitary GH4C1 cells with epidermal growth factor (EGF) caused up to a 60% reduction in the amount of [3H]MeTRH bound to specific TRH receptors. The effects of EGF were first detectable after a 2-h incubation and maximal by 24-72 h. EGF elicited a half-maximal response at 0.03 nM. Equilibrium binding analysis was performed on intact cells that had been incubated with or without 10 nM EGF for 96 h. EGF decreased the apparent number of TRH receptors (maximum binding = 0.36 vs. 0.58 pmol/mg protein for EGF-treated and control cells, respectively) without altering the apparent affinity (dissociation constant = 6.4 vs. 7.4 nM). The effects of EGF on TRH receptors were reversible. When EGF was removed from the medium, TRH receptors returned to control levels within 48 h. To assess whether the reduction of TRH receptors was functionally important, the ability of TRH to stimulate phospholipid turnover was measured in cells with a normal complement of TRH receptors and in cells that had been treated with EGF for 72 h to reduce TRH receptor density. EGF significantly blunted the ability of TRH to stimulate release of inositol phosphates from metabolically labeled cells. TRH increased inositol monophosphate accumulation 6.3-fold in control cultures and 2.0-fold in EGF-treated cells. These data show that EGF regulates the concentration of TRH receptors on pituitary GH4C1 cells and the responsiveness of the cells to TRH.     

TRH acts as a multifunctional hypophysiotropic factor in vertebrates

Thyrotropin-releasing hormone (TRH) is the first hypothalamic hypophysiotropic neuropeptide whose sequence has been chemically characterized. The primary structure of TRH (pGlu-His-Pro-NH₂) has been fully conserved across the vertebrate phylum. TRH is generated from a large precursor protein that contains multiple repeats of the TRH progenitor tetrapeptide Gln-His-Pro-Gly. In all tetrapods, TRH-expressing neurons located in the hypothalamus project towards the external zone of the median eminence while in teleosts they directly innervate the pars distalis of the pituitary. In addition, in frogs and teleosts, a bundle of TRH-containing fibers terminate in the neurointermediate lobe of the pituitary gland. Although TRH was originally named for its ability to trigger the release of thyroid-stimulating hormone (TSH) in mammals, it later became apparent that it exerts multiple, species-dependent hypophysiotropic activities. Thus, in fish TRH stimulates growth hormone (GH) and prolactin (PRL) release but does not affect TSH secretion. In amphibians, TRH is a marginal stimulator of TSH release in adult frogs, not in tadpoles, and a major releasing factor for GH and PRL. In birds, TRH triggers TSH and GH secretion. In mammals, TRH stimulates TSH, GH and PRL release. In fish and amphibians, TRH is also a very potent stimulator of α-melanocyte-stimulating hormone release. Because the intermediate lobe of the pituitary of amphibians is composed by a single type of hormone-producing cells, the melanotrope cells, it is a suitable model in which to investigate the mechanism of action of TRH at the cellular and molecular level. The occurrence of large amounts of TRH in the frog skin and high concentrations of TRH in frog plasma suggests that, in amphibians, skin-derived TRH may exert hypophysiotropic functions.     

Identification of the chicken growth hormone-releasing hormone receptor (GHRH-R) mRNA and gene: regulation of anterior pituitary GHRH-R mRNA levels by homologous and heterologous hormones

GHRH stimulates GH secretion in chickens as in mammals. However, nothing is known about the chicken GHRH receptor (GHRH-R). Here we report the cDNA sequence of chicken GHRH-R. Comparison of the cDNA sequence with the chicken genome localized the GHRH-R gene to chicken chromosome 2 and indicated that the chicken GHRH-R gene consists of 13 exons. Expression of all exons was confirmed by RT-PCR amplification of pituitary mRNA. The amino acid sequence predicted by the GHRH-R cDNA is homologous to that in other vertebrates and contains seven transmembrane domains and a conserved hormone-binding domain. The predicted size of the GHRH-R protein (48.9 kDa) was confirmed by binding of (125)I-GHRH to chicken pituitary membranes and SDS-PAGE. GHRH-R mRNA was readily detected by RT-PCR in the pituitary but not in the hypothalamus, total brain, lung, adrenal, ovary, or pineal gland. Effects of corticosterone (CORT), GHRH, ghrelin, pituitary adenylate cyclase-activating peptide, somatostatin (SRIF), and TRH on GHRH-R and GH gene expression were determined in cultures of chicken anterior pituitary cells. GHRH-R and GH mRNA levels were determined by quantitative real-time RT-PCR. Whereas all treatments affected levels of GH mRNA, only CORT, GHRH, and SRIF significantly altered GHRH-R mRNA levels. GHRH-R gene expression was modestly increased by GHRH and suppressed by SRIF at 4 h, and CORT dramatically decreased levels of GHRH-R mRNA at 72 h. We conclude that adrenal glucocorticoids may substantially impact pituitary GH responses to GHRH in the chicken through modulation of GHRH-R gene expression.     

Differential regulation of thyroid hormone receptor messenger ribonucleic acid levels by thyrotropin-releasing hormone

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.     

Regulator of G protein signaling 4 suppresses basal and thyrotropin releasing-hormone (TRH)-stimulated signaling by two mouse TRH receptors, TRH-R(1) and TRH-R(2)

We cloned the mouse TRH receptor type 2 (mTRH-R2) gene, which is 92% identical with rat TRH-R2 and 50% identical with mTRH-R1 at the amino acid level, and identified an intron within the coding sequence that is not present in the TRH-R1 gene structure. Similar to its rat homolog, mTRH-R2 binds TRH with an affinity indistinguishable from mTRH-R1, signals via the phosphoinositide pathway like mTRH-R1, but exhibits a higher basal signaling activity than mTRH-R1. We found that regulator of G protein signaling 4 (RGS4), which differentially inhibits signaling by other receptors that couple to Gq, inhibits TRH-stimulated signaling via mTRH-R1 and mTRH-R2 to similar extents. In contrast, other RGS proteins including RGS7, RGS9, and GAIP had no effect on signaling by mTRH-R1 or mTRH-R2 demonstrating the specificity of RGS4 action. Interestingly, RGS4 markedly inhibited basal signaling by mTRH-R2. Inhibition of basal signaling of mTRH-R2 by RGS4 suggests that modulation of agonist-independent signaling may be an important mechanism of regulation of G protein-coupled receptor activity under normal physiologic circumstances.