No abstract

Cortisol secretion in adrenal Cushing's syndrome can be regulated by aberrant hormone receptors, such as gastric inhibitory polypeptide, V1 vasopressin, catecholamines, LH/human chorionic gonadotropin, and serotonin receptors. We report the case of a patient with Cushing's syndrome due to bilateral adrenal macronodular hyperplasia. Extensive in vivo testing for the presence of aberrant receptors revealed a 5-fold increase of plasma cortisol after the administration of cisapride, an agonist of the serotonin 4 (5-HT(4)) receptor. Primary cell cultures were established from adrenocortical specimens obtained at surgery, and in vitro studies also showed that cisapride determined an increase [133.7 +/- 5.5% (mean +/- SE) of baseline, considered 100%) of cortisol secretion from cultured cells. The presence of 5-HT(4) receptor transcript, and in particular of isoforms c, g, and n, was confirmed by RT-PCR, and the determination of the mRNA levels by real-time RT-PCR revealed a higher expression than in normal adrenal glands. To our knowledge, this is one of the first reports of Cushing's syndrome in which cortisol secretion is regulated mainly by the 5-HT(4) receptor, among known aberrant receptors. In addition, it is noteworthy that hypocortisolism ensued after the removal of the most enlarged adrenal gland, but the in vivo response to cisapride persisted.

Objective: Reduced expression or defective targeting of the sodium/iodide symporter (NIS) to the cell membrane in thyroid tumours has been reported. The expression of the NIS gene is up-regulated by TSH through the cAMP pathway and the characterization of the promoter region of the rat NIS gene revealed the existence of a degenerate cAMP response element (CRE) sequence. The cAMPdependent transcription factor cAMP response element-binding protein (CREB) binds to CRE acting, upon phosphorylation, as a transcriptional activator. In this study we evaluated the expression of CREB and NIS gene in thyroid non-functioning adenomas ðn ¼ 18Þ and carcinomas ðn ¼ 20Þ; as well as in the corresponding normal tissue. Methods: The levels of CREB and NIS mRNA were determined by quantitative real-time RT-PCR, whereas CREB protein (total and phosphorylated) was analyzed by Western blot. Results: The levels of CREB mRNA in thyroid carcinomas, but not in adenomas, were significantly lower than in the corresponding normal tissue ð4:63^0:89 vs 9:51^2:01 pg/mg total RNA, meansŜ .E., P ¼ 0:025Þ: CREB protein levels, which were determined in a subset of samples, were in quite good agreement with mRNA data. NIS mRNA levels did not differ in adenomas or carcinomas, compared with the corresponding normal tissue and no significant relationship with the levels of CREB mRNA was observed. Conclusions: Our results have indicated for the first time that reduced levels of CREB expression are a feature of thyroid carcinomas, and confirm that different factors are likely to modulate NIS expression.

Objective: The pathogenesis of thyroid hyperfunctioning adenomas is still only partially understood and controversy exists about the frequency of gain-of-function mutations of the TSH receptor or G s a gene, which activate the cAMP pathway. The nuclear transcription factors cAMP-responsive element binding protein (CREB) and inducible cAMP early repressor (ICER) are among the final targets of this signalling cascade. Design: In our study we focused on the expression of CREB and ICER genes in the nodular as well as in the extranodular tissue of hyperfunctioning tumours of the thyroid. Methods: RT-PCR and Western blot analysis were performed in a series of 14 patients. The presence of an activating mutation of the TSH receptor or of the G s a gene was ascertained by direct sequencing. Results: The levels of CREB transcripts did not significantly differ in the adenomas and in the normal tissues (CREB/GAPDH, mean optical density^S.E.: 0:98^0:18 vs 0:88^0:27 respectively, P = not significant (N.S.)), although case-to-case variability was observed. The absence of a significant difference between the adenoma and the surrounding normal tissue was maintained after dividing the patients into two groups, according to TSH receptor status. Accordingly, no significant difference in the levels of CREB protein (total and Ser 133 -phosphorylated) was observed between the nodular and the extranodular tissue. In addition, no difference was found in the levels of ICER transcripts (ICER/GAPDH, mean optical density^S.E.: 0:52^0:11; nodule vs 0:36^0:11; normal thyroid, P ¼ N:S:), independently of the TSH receptor gene status (i.e. wild-type or mutated). Conclusions: Our results support the recent hypothesis that the activation of the cAMP pathway in hyperfunctioning adenomas of the thyroid might be counteracted by opposite events and suggest that complex molecular mechanisms might take part in the pathogenesis of hyperfunctioning tumours.

In about 30-40% of GH-secreting adenomas, gain-of-function mutations of the Gsalpha gene, which convert this gene into an oncogene termed gsp, occur. Gsalpha mutations have been related to pituitary tumorigenesis. We focused on 2 nuclear transcription factors that are final targets of the cAMP-dependent pathway and are positively regulated by cAMP signaling, i.e. the cAMP-responsive element binding protein (CREB) and the inducible cAMP early repressor (ICER), that derives from alternative splicing of cAMP-responsive element modulator gene. We examined 21 GH-secreting adenomas, 8 with (gsp(+)) and 13 without (gsp(-)) a mutated Gsalpha. Analysis of CREB and ICER I/II messenger RNA revealed that the levels of both transcripts were higher in gsp(+) than in gsp(-) tumors (CREB/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mean optical density +/- SE, 2.34 +/- 0.36 in gsp(+) vs. 0.99 +/- 0.22 in gsp(-), P = 0.003; ICER I/GAPDH, 0.53 +/- 0.15 in gsp(+) vs. 0.14 +/- 0.07 in gsp(-), P = 0.01; ICER II/GAPDH, 1.5 +/- 0.21 in gsp(+) vs. 0.83 +/- 0.13 in gsp(-), P = 0.01), although a few cases in both groups did not display this pattern of expression. Moreover, no positive correlation between the levels of CREB and ICER transcripts was observed, suggesting the possible presence of alterations in the mechanisms by which cAMP signaling directs the expression of CREB and/or ICER genes. Our results indicate a complex pattern of expression of nuclear transcription factors that mediate cAMP action in both gsp(+) and gsp(-) tumors, suggesting that, beside Gsalpha gene mutations, different and partially unknown molecular events may contribute to the pathogenesis of these tumors.