The molecular mechanisms leading to adrenocortical tumorigenesis have been only partially elucidated so far. Because the pituitary hormone ACTH, via activation of the cAMP pathway, regulates both cell proliferation/differentiation and steroid synthesis in the adrenal cortex, in this study we focused on the cAMP-dependent transcription factors cAMP responsive element modulator (CREM) and cAMP responsive element binding protein (CREB). We studied CREM and CREB expression by RT-PCR in human normal adrenal cortex (n = 3), adrenocortical adenomas (n = 8), and carcinomas (n = 8). We found transcripts corresponding to the isoforms alpha, beta, gamma, and tau2 of the CREM gene in all of the normal adrenal tissues, in the adenomas, and in seven of eight carcinomas. On the other hand, mRNA for the inducible cAMP early repressor isoforms, which derive from an internal promoter of CREM gene, was detected in the normal adrenal and in seven of eight adenomas, but in only three of eight carcinomas. Similarly, CREB transcripts were readily detectable in all normal adrenals and adenomas, whereas they were not found in four of eight adrenal carcinomas. To further characterize the carcinomas, telomerase activity and the expression of the ACTH receptor gene were determined. Telomerase activity in the carcinomas resulted in levels significantly higher than in the adenomas, whereas the levels of ACTH receptor mRNA were lower in the carcinomas. No correlation was found in the carcinomas between the levels of the ACTH receptor transcript and the loss of expression of CREB/inducible cAMP early repressor, suggesting that this alteration is not secondary to an upstream disregulation at the receptor level. In conclusion, our results suggest that an alteration in cAMP signaling may be associated with malignancies of the adrenal cortex.

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.