Regulation of the expression of the prepro-TRH (ppTRH) gene by epidermal growth factor (EGF) was investigated. The i.p. injection of EGF significantly stimulated hypothalamic ppTRH messenger RNA levels in rats. To clarify whether this stimulatory effect of EGF could be exerted at the level of gene transcription, the 5'-flanking region (-1893/+127) of the mouse ppTRH gene fused to a luciferase reporter gene was transiently transfected into pituitary GH4C1 cells, and the effect of EGF on gene transcription was measured by a luciferase assay. EGF stimulated ppTRH gene promoter activity in a time- and dose-dependent manner. Deletion analysis revealed that two different regions of the promoter, between -254 and -218 [EGF response element-1 (EGFRE1)] and between -130 and -84 (EGFRE2) were required for full stimulation by EGF. The two EGFREs possessed putative binding sequences for the transcription factor Sp1, and they functioned cooperatively in heterologous promoters. Nuclear extracts from GH4C1 cells specifically bound those two EGFREs in gel retardation assays. Two protein-DNA complexes were found on EGFRE1, whereas four complexes were observed on EGFRE2. Although the binding of nuclear extracts to EGFRE1 was competed for by the consensus Sp1 binding sequence, the complexes on EGFRE1 were not supershifted by an Sp1 antibody. Formation of the slower migrating protein complex on EGFRE1 was prevented by EDTA, suggesting that one of the EGFRE1-binding proteins might be an Sp1-related zinc finger protein. Competition and supershift experiments demonstrated that the EGFRE2-binding protein showing that the slowest migration possessed a characteristic similar to that of Sp1. Selective mutations of the Sp1-binding site in EGFRE2 markedly diminished the EGF-induced stimulation. These results suggest that EGF may function as a positive regulator of ppTRH gene expression, and that the stimulatory effect may be mediated through a cooperative interaction between Sp1 or Sp1-related proteins and additional factors that bind to two separate DNA regions.

To gain insight into the mechanism underlying the epidermal growth factor (EGF)-induced changes in responsiveness to TRH and in the numbers of TRH receptors (TRH-Rs) in the pituitary, we investigated the transcriptional regulation by EGF of the TRH-R gene in GH4C1 cells. Northern blot analyses and binding studies revealed that EGF reduced both TRH binding and TRH-R mRNA levels in a dose- and time-dependent manner, while no significant changes were observed in beta-actin mRNA levels. Addition of actinomycin D caused an acute increase in the basal TRH-R mRNA level, and the rate of decrease of the TRH-R mRNA was identical in control and EGF-treated groups, suggesting that the stability of the TRH-R mRNA was not significantly affected in EGF-treated cells. Incubation with cycloheximide also induced an increase in the basal TRH-R mRNA level and completely reversed the EGF-induced reduction of TRH-R mRNA levels. Furthermore, a nuclear run-on assay demonstrated that the rate of transcription of the TRH-R gene was significantly inhibited in cells treated with EGF. We conclude that (1) EGF decreases the expression of the TRH-R mRNA largely by reducing its rate of transcription, and this action requires the synthesis of new proteins, and (2) inhibitors of protein and RNA synthesis cause a significant increase in the basal TRH-R mRNA level, suggesting that there may be a short-lived protein suppressing the TRH-R mRNA level in the pituitary.

A response of growth hormone (GH) to thyrotropin-releasing hormone (TRH) is observed in lower mammals and patients with diseases such as a cromegaly, but not in normal subjects. We have previously demonstrated the existence of intact TRH receptor mRNA in GH-secreting adenoma. To examine whether intact somatotrophs in the anterior pituitary also express TRH receptor, we attempted to localize both TRHR mRNA and GH immunoreactivity simultaneously. In situ hybridization analysis revealed TRHR mRNAs specifically in the anterior pituitary, and 61.1% of the anterior pituitary cells expressed this transcript. Staining for GH and PRL on the same samples showed that the somatotrophs apparently expressed TRHR mRNA and approximately 62.3% and 30.9% of hybridization-positive cells were somatotorophs and mammotrophs, respectively. Moreover, TRHR mRNA level in the somatotrophs expressed as the number of silver grains per cell was equivalent to that in the mammotrophs. These findings demonstrated expression of the TRHR mRNA in somatotrophs in the rat anterior pituitary, and also showed that more than 50% of the TRHR mRNA detected in the anterior pituitary was derived from these cells.

No abstract

Human thyrotropin-releasing hormone (TRH)-associated peptide 3 (hTAP-3), one of the cryptic peptides resulting from the proteolytic processing of preproTRH to produce TRH, was measured in human plasma from normal, hyperthyroid, and hypothyroid subjects. The dilution curve of hTAP-3 immunoreactivity in the serum paralleled the standard curve of the radioimmunoassay. HPLC analysis revealed a single strong immunoreactive peak, which corresponded to the authentic peptide, hTAP-3. The half-life of hTAP-3 in serum was approximately 3.5 min, and the addition of aprotinin and EDTA completely prevented its degradation. In hyperthyroid patients, plasma concentrations of hTAP-3 were significantly higher than those in the control group and hypothyroid patients, but no correlation was found between its level and that of thyroid hormone. These findings indicate the existence of intact hTAP-3 in the human serum and increases in plasma hTAP-3 levels in hyperthyroid patients, suggesting that blood hTAP-3 may be derived from the peripheral organs rather than the hypothalamus.