No abstract

Although corticotropin-releasing hormone (CRH) plays a pivotal role in the regulation of the hypothalamo-pituitary-adrenal axis, the mechanism of CRH gene expression in the neuronal cell is not completely understood. In this study, we examined the transcriptional regulation of human CRH gene 58-promoter, using a human BE(2)C neuroblastoma cell line expressing intrinsic CRH. In particular, we focused on the involvement of calmodulin kinases (CaMKs), which are known to play an important role in excitation-induced gene expression through the rise in intracellular calcium in the central nervous system. RT-PCR analysis confirmed the expression of CaMK as well as CRH mRNA in BE(2)C cells. When we introduced <1·1 kb of the 58-promoter region of the human CRH fused with luciferase reporter gene into the cells, a substantial transcriptional activity was observed, and this was further increased by the activation of the cAMP/PKA pathway. We then examined the effect of activation of CaMKs by introducing the expression vectors of each kinase, revealing a potent stimulatory effect of CaMKIV, but no effect of CaMKII. Depolarization of the cells caused an increase in CRH promoter activity, which was completely abolished by the treatment with the CaMK antagonist K252a. Interestingly, KCREB, a dominant negative form of CREB, antagonized the effect of the CaMKIV-mediated effect. Altogether, we conclude that not only the cAMP/PKA but also the calcium/CaMKIV signaling pathway is involved in the regulation of CRH gene expression. Furthermore, CREB is thought to be involved in CaMK-as well as cAMP/PKA-mediated CRH gene expression. Since the CRH gene is expressed in the neuronal cells of the hypothalamus, the calcium/CaMKIV signaling pathway may play an important role in the excitation-mediated regulation of CRH synthesis.

We examined the role of intracellular calcium release in the regulation of CRH-induced ACTH secretion using the AtT20 corticotroph cell line. We found that ruthenium red, an inhibitor of ryanodine receptor, substantially diminished the secretory response, whereas Xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, had no effect. Expression of two ryanodine receptor subtypes (RyR1 and RyR3) was confirmed by RT-PCR. We also found that caffeine, a ryanodine receptor agonist, significantly stimulated, whereas thapsigargin, which causes depletion of intracellular calcium store, markedly diminished, the ACTH release. These results suggest that ryanodine receptor-mediated calcium-induced calcium release is involved in the regulation of CRH-induced ACTH release.

Abstract. We have developed a new test for estimating the secretory capacity of parathyroid hormone (PTH) from the parathyroid gland. Sodium bicarbonate solution [8.4% (w/v); 35 ml/m 2 body surface area] was infused for 2 min, and blood samples for the determination of plasma ionized calcium, plasma PTH (intact, midregion, carboxy-terminus) and related parameters were serially obtained. In 8 healthy volunteers, the mean (±SE) plasma ionized calcium fell promptly and significantly (from 1.21 ± 0.01 to 1.11 ± 0.01 mmol/L) after the sodium bicarbonate infusion. The mean (±SE) plasma intact PTH increased promptly and significantly, by more than four fold (42.3 ± 4.2 to 182.4 ± 34.7 pg/ml), and then gradually returned to basal levels. In patients with partial hypoparathyroidism who have detectable basal plasma levels of PTH, the absolute increment in PTH levels was much less, and in the plasma obtained from patients with complete hypoparathyroidism, absolutely no response was observed. Plasma obtained from patients diagnosed with primary hyperparathyroidism (parathyroid adenoma or hyperplasia) has high basal PTH levels. The response to the sodium bicarbonate infusion in these patients was markedly blunted (less than a two-fold increase in all cases examined). No significant adverse effects were observed during the procedure. Therefore, the sodium bicarbonate infusion test is a simple and sensitive method to stimulate PTH release, and is clinically useful for evaluating parathyroid gland function.

The V(1b) vasopressin receptor, expressed mainly in the corticotroph of the anterior pituitary, mediates the stimulatory effect of vasopressin on ACTH release. To clarify the regulation of receptor expression, we cloned, sequenced (up to approximately 5 kb from the translation start site), and characterized the 5'-flanking region of the rat V(1b) receptor gene. We identified the transcription start site by amplification of cDNA ends and found a new intron within the 5'-untranslated region (5'-UTR) by comparing the sequence with that of cDNA. We then confirmed that the obtained promoter indeed has transcriptional activity by use of the luciferase reporter in AtT-20 mouse corticotroph cells. Interestingly, there were five short upstream open reading frames (uORFs) located within the 5'-UTR that were found to suppress V(1b) expression. Subsequent mutational analyses showed that the two downstream uORFs have an inhibitory effect on expression in both homologous and heterologous contexts. Furthermore, the inhibition did not accompany a parallel decrease in mRNA, suggesting that the suppressive effect occurs at a level downstream of transcription. Taken together, our data strongly suggest that the expression of the V(1b) receptor is regulated at the posttranscriptional as well as transcriptional level through uORFs within the 5'-UTR region of the mRNA. Whether the uORF-mediated regulation of V(1b) expression is functionally linked to any intracellular and/or extracellular factor(s) awaits further research.