Trh Genes Research Articles

Thyrotropin-releasing hormone gene expression in the anterior pituitary. IV. Evidence for paracrine and autocrine regulation.

Disulfiram (Dis), an inhibitor of peptidyl-glycine alpha-amidating monooxygenase, the enzyme responsible for the production of alpha-amidated peptides from their immediate, glycine-extended precursors was used to investigate the paracrine effects of TRH on anterior pituitary (AP) hormone secretion. It reduces the production of TRH without directly affecting the classical pituitary hormones, none of which is amidated. Dis (8 microM) decreased the accumulation of TRH accompanied by an equimolar increase in TRH-Gly levels, indicating that pro-TRH biosynthesis persisted. TRH and TSH release into the medium was significantly lowered, whereas other pituitary hormones were unaffected. In contrast, dexamethasone (10 nM), which up-regulates TRH gene expression in this system, increased TRH (+89.5%) and TSH (+61.3%) secretion. The combination of dexamethasone and Dis further diminished the release of TRH (-73%) and TSH (-40.3%) observed with Dis alone, indicating that TRH synthesized within the AP regulates TSH secretion. Dis significantly elevated prepro-TRH (25-50) and pro-TRH messenger RNA levels, suggesting that reduced TRH formation leads to increased pro-TRH biosynthesis and that TRH regulates its own secretion. Thus, TRH synthesized by cultured AP cells not only stimulates TSH release through a paracrine effect, but has a negative feedback on its own biosynthesis by an autocrine mechanism.

Thyrotropin-Releasing Hormone Gene Expression in the Anterior Pituitary. IV. Evidence for Paracrine and Autocrine Regulation

Disulfiram (Dis), an inhibitor of peptidyl-glycine α-amidating monooxygenase, the enzyme responsible for the production ofα -amidated peptides from their immediate, glycine-extended precursors was used to investigate the paracrine effects of TRH on anterior pituitary (AP) hormone secretion. It reduces the production of TRH without directly affecting the classical pituitary hormones, none of which is amidated. Dis (8 μm) decreased the accumulation of TRH accompanied by an equimolar increase in TRH-Gly levels, indicating that pro-TRH biosynthesis persisted. TRH and TSH release into the medium was significantly lowered, whereas other pituitary hormones were unaffected. In contrast, dexamethasone (10 nm), which up-regulates TRH gene expression in this system, increased TRH (+89.5%) and TSH (+61.3%) secretion. The combination of dexamethasone and Dis further diminished the release of TRH (−73%) and TSH (−40.3%) observed with Dis alone, indicating that TRH synthesized within the AP regulates TSH secretion. Dis significantly elevated prepro-TRH (25–50) and pro-TRH messenger RNA levels, suggesting that reduced TRH formation leads to increased pro-TRH biosynthesis and that TRH regulates its own secretion. Thus, TRH synthesized by cultured AP cells not only stimulates TSH release through a paracrine effect, but has a negative feedback on its own biosynthesis by an autocrine mechanism.

Glucocorticoids modulate the biosynthesis and processing of prothyrotropin releasing-hormone (proTRH).

The thyrotropin- (TRH) releasing hormone precursor (26 kDa) undergoes proteolytic cleavage at either of two sites, generating N-terminal 15 kDa/9.5 kDa or C-terminal 16.5/10 kDa intermediate forms that are processed further to yield five copies of TRH-Gly and seven non-TRH peptides. Glucocorticoids (Gcc) have been shown to enhance TRH gene expression in three different cell systems in vitro, an effect that occurs, at least in part, through transcriptional activation. Although this implies that an increase of TRH prohormone biosynthesis would take place, this had not been demonstrated as yet. We report here that the synthetic glucocorticoid dexamethasone (Dex) substantially elevated the de novo biosynthesis of the intact 26-kDa TRH prohormone and its intermediate products of processing in cultured anterior pituitary cells, an observation that is consistent with an overall upregulation of both the biosynthesis and degradation of the TRH precursor. We reasoned that Gcc may act not only at the transcriptional, but also at the translational/posttranslational level. To address this question we chose a different cell system, AtT20 cells transfected with a cDNA encoding preproTRH. Since TRH gene expression in these cells is driven by the CMV-IE promoter and not by an endogenous "physiological" promoter, these cells provide an ideal model to study selectively the effects of Gcc on the translation and posttranslational processing of proTRH without interference from a direct transcriptional activation of the TRH gene. Dex caused a significant 75.7% increase in newly synthesized 26-kDa TRH prohormone, suggesting that the glucocorticoid raised the translation rate. We then demonstrated that Dex treatment accelerated TRH precursor processing. Of interest, processing of the N- vs the C-terminal intermediate was influenced differentially by the glucocorticoid. Although the N-terminal intermediate product of processing accumulated, the C-terminal intermediate was degraded more rapidly. Consistent with these observations was the finding that the intracellular accumulation of the N-terminally derived peptide preproTRH 25-50 was enhanced, but levels of the C-terminally derived peptide preproTRH208-255 were reduced. Accumulation of TRH itself, whose five copies are N- and C-terminally derived, was also enhanced. We conclude that Gcc induce changes in the biosynthesis and processing of proTRH by increasing the translation rate and by differentially influencing the processing of N- vs C-terminal intermediates of the precursor molecule. These effects of Gcc at the translational and posttranslational levels result in an increase in TRH production accompanied by differential effects on the accumulation of N- and C-terminal non-TRH peptides.

Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta, India, and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan.

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, was initiated in January 1994. The incidence of cases of V. parahaemolyticus infection suddenly increased in February 1996 and has remained high since then. One hundred thirty-four strains of V. parahaemolyticus isolated from January 1994 to August 1996 were examined for serovar, the presence of the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin genes (trh1 and trh2), production of urease, and antibiogram. Strains of the O3:K6 serovar appeared for the first time in February 1996. The O3:K6 serovar strains accounted for 50 to 80% of the strains isolated during the high-incidence period (February to August 1996). All of the serovar O3:K6 strains carried the tdh gene but not the trh genes and did not produce urease. All of the isolates except two were sensitive to all of the antibiotics tested. These and the results of analysis by an arbitrarily primed PCR method indicated that the O3:K6 serovar strains belong to a unique clone. When the O3:K6 serovar strains, isolated from travelers arriving in Japan from Southeast Asian countries, were compared by the arbitrarily primed PCR method, the strains isolated between 1982 and 1993 were distinct from Calcutta O3:K6 while the strains isolated in 1995 and 1996 were indistinguishable from the Calcutta O3:K6 strains. The results suggest that this unique O3:K6 clone may have become prevalent not only in Calcutta but also in Southeast Asian countries very recently. Not only the O3:K6 strains but also the non-O3:K6, tdh-bearing strains isolated in 1996 produced thermostable direct hemolysin at high levels, and thus the level of hemolysin produced does not appear to have influenced the high incidence of serovar O3:K6 strains.

Analysis of the thermostable direct hemolysin (tdh) gene and the tdh-related hemolysin (trh) genes in urease-positive strains of Vibrio parahaemolyticus isolated on the West Coast of the United States.

Urease-positive (Ure+) and urease-negative (Ure-) strains of Vibrio parahaemolyticus isolated from patients on the West Coast of the United States between 1979 and 1995 were analyzed for the thermostable direct hemolysin (tdh) gene and the tdh-related hemolysin (trh) genes (trh1 and trh2). The DNA colony hybridization method with the polynucleotide probes was used to determine the distribution of the genes. Of 60 Ure+ strains, 59 strains (98%) had the trh (either trh1 or trh2) gene and 54 strains (90%) carried the tdh gene. The absence of the trh gene or a related sequence in an exceptional Ure+ strain was confirmed by Southern blot analyses. The stronger correlation with the trh gene than with the tdh gene was mostly attributable to strains possessing only the trh2 gene. Of 25 Ure- strains, 20 strains (80%) had the tdh gene but none had the trh gene. These results indicate a very strong correlation between the Ure+ phenotype and the trh gene and are consistent with those reported for strains isolated in Asia. The Ure+ strains carrying the trh genes were not restricted to a unique group of the strains. The O4:K12 strains carrying the trh1 gene have predominantly been isolated since 1979. However, strains of various non-O4:K12 serovars carrying either the trh1 or the trh2 gene became predominant after 1992. In addition, analysis by the arbitrarily primed PCR method revealed two subgroups within the selected Ure+ O4:K12 strains. Hybridization tests with oligonucleotide probes demonstrated that the trh1 sequences of the West Coast strains differ to some extent from those of Asian strains. Nevertheless, a PCR method previously established to detect both the trh1 and the trh2 genes in Asian strains could detect 98% of those genes in the West Coast strains.

Evidence for genetic linkage between the ure and trh genes in Vibrio parahaemolyticus.

Although V. parahaemolyticus does not generally produce urease, several studies have reported urease-positive V. parahaemolyticus isolates from clinical sources. Recently, studies have shown a complete coincidence between the urease-producing phenotype of V. parahaemolyticus strains and the possession of the thermostable direct haemolysin (TDH)-related haemolysin (TRH) gene (trh). TRH, like TDH, is considered to be an important virulence factor in the pathogenesis of V. parahaemolyticus gastroenteritis. The present study attempted to identify the gene ure encoding urease in V. parahaemolyticus to clarify the relationship between urease production and possession of trh. The polymerase chain reaction with mixed oligonucleotide primers targeted for conserved sequences of reported ure genes from other species was used to prepare a DNA probe to detect the V. parahaemolyticus ure gene. Colony hybridisation with this ure probe demonstrated that all the ure-positive strains produced urease. Considering the coincidence between production of urease and possession of trh in V. parahaemolyticus, it was concluded that the presence or absence of the ure gene is completely coincident with that of the trh gene in V. parahaemolyticus strains. Furthermore, the relative location of ure and trh on V. parahaemolyticus chromosomal DNA was analysed by pulsed-field gel electrophoresis. The results showed that, in all the strains examined, ure and trh were detected on the same NotI fragment, showing that the two genes localise within a relatively small portion of the chromosome DNA. These results suggest that the ure and trh genes are genetically linked in V. parahaemolyticus strains.

Thyrotropin-releasing hormone gene expression by anterior pituitary cells in long-term cultures is influenced by the culture conditions and cell-to-cell interactions.

It has been suggested that TRH, synthesized by anterior pituitary (AP) cells in long-term monolayer cultures, may act as a paracrine or autocrine regulator. Because local control through messenger molecules depends on the cellular microenvironment, we were interested in studying the synthesis of TRH by AP cells in different culture systems and under various conditions. When AP cells were cultured as monolayers in medium containing 10% FCS for long periods of time (up to 3 weeks), a considerable increase in TRH content and prepro-TRHmessengerRNA (preproTRHmRNA) levels could be demonstrated by RIA and Northern blot analysis, whereas the cellular content of the TRH-like peptide pyroGlu-Glu-Pro-NH2 decreased with time in culture to undetectable levels. The release of TRH could be stimulated by depolarizing concentrations of K+ (55 mM), by the Ca++ ionophore A23187, and by GnRH, but not by CRH or GRF, indicating that TRH is stored in gonadotropes. Moreover, a combined in situ hybridization and immunocytochemical analysis demonstrated colocalization of LH in preproTRHmRNA-positive AP cells. When AP cells were cultured as reaggregates in the same (FCS-containing) medium, only a marginal increase in TRH content and preproTRHmRNA levels was observed. Irrespective of the culture systems and the culture conditions used, TRH gene expression was not observed when FCS was omitted. These results indicate that TRH gene expression more likely reflects derepression, rather than induction, of the TRH gene.

Urea hydrolysis and suppressed production of thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus associated with presence of TDH-related hemolysin genes.

A total of 18 strains of V. parahaemolyticus isolated from patients of past food poisoning cases occurring in Kanagawa Prefecture, Japan, were assayed for presence of the thermostable direct hemolysin (TDH) gene and the TDH-related hemolysin (TRH) genes (trh 1 and trh 2) with specific reference to their ability to hydrolyze urea and TDH production. A polymerase chain reaction assay revealed that all urea-hydrolyzing strains (9 strains) carried either trh 1 gene or trh 2 gene. The strains carrying the trh genes as well as the tdh gene produced TDH less by a factor of 4 to 16 than those carrying only the tdh gene, suggesting the expression of the tdh gene was suppressed by the presence of trh gene through a mechanism yet to be defined.

Levels of thermostable direct hemolysin production by Vibrio parahaemolyticus strains carrying both tdh and trh genes

One hundred and twenty-five strains of Vibrio parahaemolyticus carrying both the tdh and trh genes were selected from the strains isolated from the travelers with diarrhea by an hybridization test using polynucleotide probes. The levels of TDH produced by these strains and the association between the TDH levels and related characteristics in these strains were analyzed. The TDH level varied greatly from strain to strain, but none of the levels was as high as that of the typical Kanagawa phenomenon-positive strains. The strains were classified into "TDH producer" (18 strains), "Low-level TDH producer" (85 strains), and "No TDH producer" (22 strains) based on the results of a modified Elek test and the hemolysis assay on Wagatsuma agar. The highest TDH level achieved by the "TDH producer" was twofold lower than that of the Kanagawa phenomenon-positive strains as assayed by the RPLA method. All strains possessed the toxR gene. The trh1 and trh2 genes were detected in, respectively, 105 and 20 strains, and no correlation existed between the type of the trh gene and the levels of TDH produced. Considerable restriction fragment length polymorphism was observed with the tdh gene-bearing HindIII DNA fragment in different strains, but it was not related with the TDH level.

Incidence of Kanagawa phenomenon-positive and -negative Vibrio parahaemolyticus strains isolated from traveller's diarrhea and their relation to tdh and trh genes

A total of 1,319 strains of Vibrio parahaemolyticus isolated from traveller's diarrhea were analysed for Kanagawa phenomenon (KP) with the Wagatsuma blood agar test and the results were also compared with those of analyses of tdh and trh genes which encode thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). The majority of the strains (1,152 strains) counting 87.3% had positive KP, among which 1,049 and 103 strains were only tdh and both tdh and trh-positive ones, respectively. However, 167 strains counting 12.7%, which is quite high compared to the previous report, were found to have negative KP, among which 94 and 24 strains were only trh and both tdh and trh-positive ones, respectively.

Regulation of the human trh (htrh) gene by human thyroid hormone receptor β1 (hTRβ1) mutants

TRH is negatively regulated by T3 both in the hypothalamic paraventricular nucleus and transient transfection models. Mutations in hTRβ1 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) hTRβ1, and three qualitatively distinct hTRβ1 mutant forms (ED, OK and PV). In the presence of T3 (10−9 M), liganded WT-hTRβ1 inhibited hTRH promoter activity significantly (40%). Co-transfection 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 hTRβ1-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 hTRβ1 mediated inhibition, likely contribute to elevated TSH values observed in the syndrome of thyroid hormone resistance.

Quantification of rat pre-pro-thyrotropin releasing hormone (TRH) mRNA by reverse transcription-polymerase chain reaction using external and internal standardisation

We have developed a quantitative reverse transcription polymerase chain reaction (RT-PCR) method, using both internal and external standardisation, to quantitate fg amounts of pre-pro-thyrotropin releasing hormone (pre-pro-TRH) mRNA in the paraventricular nucleus (PVN) of individual laboratory rats. A constant amount of internal standard is coamplified with both the cDNA of each unknown specimen and a dilution series of an identical RT-PCR generated external standard, allowing quantitation of the samples by interpolation from the external standard curve. Pre-pro-TRH mRNA levels in the PVN were reduced by thyroxine (T 4) treatment to 48% of those in control animals, and were increased by thyroidectomy to 155% of the control levels. By combining external and internal standardisation, our method allows multiple samples and treatment groups to be assayed concurrently, thereby eliminating inter-assay variability, whilst retaining the advantages of internal standardisation. It will facilitate further studies of the control of TRH gene expression in pathophysiological conditions.

Activation of thyrotropin-releasing hormone gene expression in cultured fetal diencephalic neurons by differentiating agents.

The present studies were undertaken to investigate the effect of 5-bromo-2'-deoxyuridine (BrdU; 50 microM) or forskolin/3-isobutyl-1-methylxanthine (F/I; 10/500 microM) on TRH gene expression in cultured fetal diencephalic cells. BrdU as well as drugs such as F/I that raise intracellular cAMP levels had been previously termed differentiating agents because they cause morphological and functional differentiation of IMR-32 neuroblastoma cells. We postulated that neurons of fetal diencephalons may remain relatively undifferentiated in vitro and that this might be the reason for low or undetectable TRH production. We hypothesized that treatment with differentiating agents might increase neuropeptide expression. Both BrdU and F/I dramatically (P < 0.01) raised intracellular TRH and pro-TRH messenger RNA concentrations in cultured diencephalic neurons. Although a short BrdU exposure during the first 4 days of culture was sufficient to irreversibly change TRH neurons and to cause maintenance of high TRH levels after withdrawal of the drug, F/I had to be present continuously throughout the observation period of 16 days to significantly elevate TRH expression. This suggests that BrdU and F/I act at different intracellular sites to activate TRH expression in cultured diencephalic neurons. The reduction of glial cells that occurs concurrent with the BrdU treatment was not observed after F/I exposure, and therefore, this effect does not appear to be a key factor for the induction of TRH expression. As the intracellular accumulation of somatostatin and arginine vasopressin, which were determined in parallel, was similarly enhanced after treatment with BrdU or F/I, our culture system might provide a valuable tool for the study of these and possibly other neuropeptides in vitro.

Detection of the trh gene in Vibrio parahaemolyticus isolated from overseas travellers' diarrhea and their biochemical characteristics

A total of 478 Vibrio parahaemolyticus strains isolated from overseas travellers' diarrhea during the last 7 years of 1989 to 1995 were examined for the production of Kanagawa hemolysin by reversed passive latex agglutination (RPLA) test. Three hundred-seventy (77.4%) out of 478 strains were positive for Kanagawa hemolysin, whereas 108 strains were weakly positive or negative. For those Kanagawa hemolysin-weakly positive or negative strains, the tdh and trh genes associated with the production of TDH (thermostable direct hemolysin) and TRH (TDH-related hemolysin), respectively, were studied by the polymerase chain reaction method. The trh gene was detected in 98 (90.7%) out of 108 strains. In 35 strains belonging to 13 serotypes such as O3: K6, O1:K33, O3:K59, the trh gene alone was detected. On the other hand, both trh and tdh genes were detected in 63 strains of 17 serotypes including O1:K69, O3:K72, O6:K46. Among the strains of 4 serotypes including O1:K56, O1:KUT, O3:KUT and O5:KUT, two types of the trh positive alone and the trh and tdh positive were observed. Of interest, all of the 98 trh-positive strains were positive for the urease hydrolysis, whereas all Kanagawa hemolysin-positive strains were not. Furthermore, the strains of serotype O6:K18 (4 strains) were positive for the fermentation of dulcitol, and the strains of serotype O1:K1 (5 strains) were indole negative. These characteristics of the strains were completely different from those of typical V. parahaemolyticus strains.

Tubulogenesis in Drosophila: a requirement for the trachealess gene product.

The trachealess (trh) gene of Drosophila is required for embryonic tube formation. In trh mutants, tube-forming cells of the salivary gland, trachea, and filzkörper fail to invaginate to form tubes and remain on the embryo surface. We identified a P-element insertion that disrupts trh function and used the insert to clone and characterize trh. trh is expressed in the salivary duct, trachea, and filzköper primordia, and expression persists in these cells throughout embryogenesis. trh expression in the salivary duct is controlled by the homeotic gene, Sex combs reduced (Scr), and by another salivary gland gene, fork head (fkh). trh is homologous to two transcription factors: the human hypoxia-inducible factor-1 alpha and the Drosophila Single-minded protein.

Glucocorticoids stimulate thyrotropin-releasing hormone gene expression in cultured hypothalamic neurons.

Although there is much evidence indicating that glucocorticoids (GC) inhibit the hypothalamic-pituitary-thyroid axis in both rat and man in vivo, there have been no previous studies on the direct effect of GC on hypothalamic TRH neurons in vitro. In this laboratory, we developed fetal rat (day 17) diencephalic neuronal cultures in the presence of 5'-bromo-2-deoxyuridine, a cell-differentiating agent that stimulates TRH gene expression. In 12 separate experiments, dexamethasone (Dex) induced a 2.2-fold increase in TRH content vs. the control value (P < 0.01). Dex (10(-8)M) enhanced TRH messenger RNA (mRNA) 1.6-fold (n = 75 wells; P < 0.01) by nonisotopic in situ hybridization. On Northern blot analysis using a 32P-labeled complementary RNA probe, TRH mRNA was enhanced 3-fold (n = 4; P < 0.01). Nuclear run-on analysis revealed that Dex enhanced transcription 7.7 fold (n = 3; P < 0.01). We conclude that 1) Dex stimulates the expression of TRH peptide and TRH mRNA in cultured hypothalamic neurons; 2) the increase in TRH mRNA results (at least in part) from enhanced transcription; and 3) the reported in vivo depression of TRH in the paraventricular nucleus after GC stimulation suggests that this effect must be mediated indirectly on the TRH neuron.

Glucocorticoids stimulate thyrotropin-releasing hormone gene expression in cultured hypothalamic neurons

Although there is much evidence indicating that glucocorticoids (GC) inhibit the hypothalamic-pituitary-thyroid axis in both rat and man in vivo, there have been no previous studies on the direct effect of GC on hypothalamic TRH neurons in vitro. In this laboratory, we developed fetal rat (day 17) diencephalic neuronal cultures in the presence of 5'-bromo-2-deoxyuridine, a cell-differentiating agent that stimulates TRH gene expression. In 12 separate experiments, dexamethasone (Dex) induced a 2.2-fold increase in TRH content vs. the control value (P < 0.01). Dex (10(-8)M) enhanced TRH messenger RNA (mRNA) 1.6-fold (n = 75 wells; P < 0.01) by nonisotopic in situ hybridization. On Northern blot analysis using a 32P-labeled complementary RNA probe, TRH mRNA was enhanced 3-fold (n = 4; P < 0.01). Nuclear run-on analysis revealed that Dex enhanced transcription 7.7 fold (n = 3; P < 0.01). We conclude that 1) Dex stimulates the expression of TRH peptide and TRH mRNA in cultured hypothalamic neurons; 2) the increase in TRH mRNA results (at least in part) from enhanced transcription; and 3) the reported in vivo depression of TRH in the paraventricular nucleus after GC stimulation suggests that this effect must be mediated indirectly on the TRH neuron.

Thermostable direct hemolysin gene of Vibrio parahaemolyticus: a virulence gene acquired by a marine bacterium.

evidence for the importance of TDH in the enteropathogenicity of V. parahaemolyticus has only recently been obtained by using genetically altered strains and more-sensitive assays. The molecular studies of the gene encoding TDH (tdh) have also revealed other interesting features regarding sequence divergence, mobility, and regulation. In this review, we summarize the results of these genetic studies of the tdh gene which indicate that TDH is a major virulence determinant of KP-positive V. parahaemolyticus strains and that the KP-positive phenotype results from highlevelexpressionofaparticulartdhgene.Theseattributesmake the KP phenotype a good marker for a virulent strain. We also speculate about the origin and variation of the tdh gene and about why the virulence genes, thetdhandtdh-related hemolysin (trh) genes, are present in only a small population of V. parahaemolyticus.Theextensivedataavailableforthetdhgene help us to better understand potentially pathogenic bacteria such asV. parahaemolyticusin the natural environment.

Thyrotropin-releasing hormone and c-fos/c-jun genes are colocalized in rat anterior pituitary cells: stimulation of transcription by glucocorticoids.

We recently reported that glucocorticoids (GC) enhance the level of TRH peptide and messenger RNA in anterior pituitary cells. However, the regulating mechanism is as yet unclear. The protooncogenes c-fos/c-jun belong to the class of immediate early genes that are activated in neurons by a variety of stimuli, including GC. Fos protein acts as an intracellular third messenger, regulating gene transcription of neurotransmitters. To determine whether c-fos/c-jun are involved in regulating the effect of GC on TRH in rat anterior pituitary cells, the coexpression and nuclear transcription activity of TRH and c-fos/c-jun after dexamethasone (DEX) stimulation (7 days) were investigated. The double labeled in situ hybridization results demonstrated that TRH and c-fos/c-jun are coexpressed in anterior pituitary cells and that DEX (10(-8) M) enhanced the cell intensity for TRH and c-fos/c-jun. The mean cell intensity of treatment vs. control was 2.4-fold for TRH, 1.4-fold for c-fos, and 1.4-fold for c-jun (n = 24; P < 0.01). The Northern blot analysis also showed that DEX increased the messenger RNA level of TRH 5.1-fold (n = 4; P < 0.01), that of c-fos 1.8-fold (n = 5; P < 0.01), and that of c-jun 4.2-fold (n = 4; P < 0.01). The nuclear run-on analysis indicated that DEX increased the nuclear transcription activity of TRH 3.3-fold, that of c-jun 3.2-fold, and that of c-fos 3-fold (n = 3; P < 0.01) vs. the control value. The coexpression of TRH and c-fos/c-jun in anterior pituitary cells as well as the enhancement of transcription after DEX treatment raise the possibility that c-fos/c-jun could mediate the effect of GC on TRH gene transcriptional activity.

Thyrotropin-releasing hormone and c-fos/c-jun genes are colocalized in rat anterior pituitary cells: stimulation of transcription by glucocorticoids

We recently reported that glucocorticoids (GC) enhance the level of TRH peptide and messenger RNA in anterior pituitary cells. However, the regulating mechanism is as yet unclear. The protooncogenes c-fos/c-jun belong to the class of immediate early genes that are activated in neurons by a variety of stimuli, including GC. Fos protein acts as an intracellular third messenger, regulating gene transcription of neurotransmitters. To determine whether c-fos/c-jun are involved in regulating the effect of GC on TRH in rat anterior pituitary cells, the coexpression and nuclear transcription activity of TRH and c-fos/c-jun after dexamethasone (DEX) stimulation (7 days) were investigated. The double labeled in situ hybridization results demonstrated that TRH and c-fos/c-jun are coexpressed in anterior pituitary cells and that DEX (10(-8) M) enhanced the cell intensity for TRH and c-fos/c-jun. The mean cell intensity of treatment vs. control was 2.4-fold for TRH, 1.4-fold for c-fos, and 1.4-fold for c-jun (n = 24; P < 0.01). The Northern blot analysis also showed that DEX increased the messenger RNA level of TRH 5.1-fold (n = 4; P < 0.01), that of c-fos 1.8-fold (n = 5; P < 0.01), and that of c-jun 4.2-fold (n = 4; P < 0.01). The nuclear run-on analysis indicated that DEX increased the nuclear transcription activity of TRH 3.3-fold, that of c-jun 3.2-fold, and that of c-fos 3-fold (n = 3; P < 0.01) vs. the control value. The coexpression of TRH and c-fos/c-jun in anterior pituitary cells as well as the enhancement of transcription after DEX treatment raise the possibility that c-fos/c-jun could mediate the effect of GC on TRH gene transcriptional activity.