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Pecori Giraldi F.,University of Milan | Pecori Giraldi F.,Neuroendocrinology Research Laboratory | Ambrogio A.G.,Neuroendocrinology Research Laboratory
Endocrine | Year: 2015

The progress in assay methodology, from the use of radioactive tracers to chemiluminescent signals, from competitive to chromatographic techniques and from serum or urine to saliva has considerably impacted on hormonal measurements. The clinician now may choose among multiple tests but the inherent variability in cortisol and ACTH secretion, coupled to lack of harmonization among assay procedures and normal ranges mandates careful interpretation of any result. The present review will examine factors which affect interpretation of cortisol and ACTH measurements and their impact on tests used for management of Cushing’s syndrome. © 2015, The Author(s).

Cassarino M.F.,Neuroendocrinology Research Laboratory | Sesta A.,Neuroendocrinology Research Laboratory | Pagliardini L.,Neuroendocrinology Research Laboratory | Losa M.,Ospedale San Raffaele IRCCS | And 4 more authors.
Pituitary | Year: 2014

Purpose: It is well known that methylation plays an important role in regulating tissue expression of proopiomelanocortin (POMC) and recent studies have shown that demethylation can occur also in vitro in neuroendocrine tumors. Aim of the present study was to evaluate whether inhibition of methylation modulates POMC expression and ACTH secretion by human corticotrope tumors. Methods: Twenty two ACTH-secreting pituitary tumors were incubated with 5-AZA-2′-deoxycytidine (AZA), an inhibitor of DNA-methyltransferases, with or without 10 nM corticotropin-releasing hormone (CRH). Both dose response (100 nM–10 μM AZA) and time course (4–96 h) experiments were carried out for measurement of ACTH secretion and POMC gene expression. Results: Incubation with AZA increased constitutive POMC expression and ACTH secretion by human corticotrope adenomas. The effect appeared most notable at 24 and 48 h with 1 μM AZA. Incubation with AZA did not exert an additional stimulatory effect on CRH-stimulated POMC and ACTH. Conclusions: The present study shows that AZA increases POMC gene expression and ACTH secretion in human pituitary ACTH-secreting tumors. This can be taken to indicate that mechanisms set into motion by AZA play a role in the regulation of ACTH secretion/POMC expression in tumoral corticotropes and paves the way to further studies in Cushing’s disease. © 2013, Springer Science+Business Media New York.

Cassarino M.F.,Neuroendocrinology Research Laboratory | Sesta A.,Neuroendocrinology Research Laboratory | Pagliardini L.,Neuroendocrinology Research Laboratory | Losa M.,Ospedale San Raffaele IRCCS | And 4 more authors.
Endocrine | Year: 2016

ACTH-secreting pituitary tumors are by definition partially autonomous, i.e., secrete ACTH independent of physiological control. However, only few, small-sized studies on proopiomelanocortin (POMC) and its regulation by corticotropin-releasing hormone (CRH) or glucocorticoids are available. Objective of the present study was to report on constitutive and CRH- and dexamethasone-regulated POMC, CRH (CRH-R1), and glucocorticoid receptor (NR3C1) gene expression in a large series of human corticotrope adenomas. Fifty-three ACTH-secreting adenomas were incubated with 10 nM CRH or 10 nM dexamethasone for 24 h. POMC, CRH-R1,NR3C1, and its alpha and beta isoforms were quantified and medium ACTH measured. Constitutive POMC expression proved extremely variable, with macroadenomas exhibiting higher levels than microadenomas. POMC increased during CRH in most specimens; conversely, changes induced by dexamethasone were varied, ranging from decrease to paradoxical increase. No correlation between POMC and ACTH was detected in any experimental condition. CRH-R1 expression was not linked to the response to CRH while NR3C1 was expressed at greater levels in specimens who failed to inhibit during dexamethasone; glucocorticoid receptor α was the more abundant isoform and subject to down-regulation by dexamethasone. Our results demonstrate a considerable variability in POMC expression among tumors and no correlation between POMC and ACTH, suggesting that POMC peptide processing/transport plays a major role in modulating ACTH secretion. Further, CRH-R1 and NR3C1 expression were not linked to the expected ligand-induced outcome, indicating that receptor signaling rather than abundance determines corticotrope responses. Our findings pave the way to new avenues of research into Cushing’s disease pathophysiology. © 2016 The Author(s)

Isidori A.M.,University of Rome La Sapienza | Graziadio C.,University of Rome La Sapienza | Paragliola R.M.,Catholic University of the Sacred Heart | Cozzolino A.,University of Naples Federico II | And 4 more authors.
Journal of Hypertension | Year: 2015

Cushing's syndrome is associated with increased mortality, mainly due to cardiovascular complications, which are sustained by the common development of systemic arterial hypertension and metabolic syndrome, which partially persist after the disease remission. Cardiovascular diseases and hypertension associated with endogenous hypercortisolism reveal underexplored peculiarities. The use of exogenous corticosteroids also impacts on hypertension and cardiovascular system, especially after prolonged treatment. The mechanisms involved in the development of hypertension differ, whether glucocorticoid excess is acute or chronic, and the source endogenous or exogenous, introducing inconsistencies among published studies. The pleiotropic effects of glucocorticoids and the overlap of the several regulatory mechanisms controlling blood pressure suggest that a rigorous comparison of invivo and in-vitro studies is necessary to draw reliable conclusions. This review, developed during the first 'Altogether to Beat Cushing's syndrome' workshop held in Capri in 2012, evaluates the most important peculiarities of hypertension associated with CS, with a particular focus on its pathophysiology. A critical appraisal of most significant animal and human studies is compared with a systematic review of the few available clinical trials. A special attention is dedicated to the description of the clinical features and cardiovascular damage secondary to glucocorticoid excess. On the basis of the consensus reached during the workshop, a pathophysiology-oriented therapeutic algorithm has been developed and it could serve as a first attempt to rationalize the treatment of hypertension in Cushing's syndrome. © 2014 Wolters Kluwer Health.

Occhi G.,University of Padua | Regazzo D.,University of Padua | Albiger N.M.,University of Padua | Ceccato F.,University of Padua | And 12 more authors.
Endocrinology | Year: 2014

Cushing's disease (CD) is a rare condition in which hypercortisolemia is secondary to excessive ACTH release from a pituitary corticotroph adenoma. CD is associated with significant morbidity and mortality,anda safe therapy that effectively targets the pituitary tumor is still lacking. Retinoic acid (RA) and dopamine agonists (DAs) have recently been considered as monotherapy in CD patients, and satisfactory results have been reported, albeit in a limited number of patients. Given the permissive role of RA on the dopamine receptor type-2 (DRD2), the aim of the present study was to see whether a combination of 9-cis RA and theDAbromocriptine (Br) might represent a possible treatment for CD. Here we show that 9-cis RA induces a functional DRD2 in the pituitary corticotroph cell line AtT20, and increases cell sensitivity to Br via a mechanism only partially related to corticotroph-to-melanotroph transdifferentiation. In addition, 9-cisRAand Br act synergistically to modulate cell viability, with favorable implications for clinical use. In nearly 45% of corticotropinoma-derived primary cultures, the combined administration of 9-cis RA and Br lowered the steady-state level of the ACTH precursor proopiomelanocortin (POMC) more efficiently than either of the drugs alone. In conclusion, the effects of a combination of 9-cis RA and Br on ACTH synthesis/secretion and cell viability in AtT20, and on POMC transcriptional activity in human corticotropinomas might represent a suitable starting point for assessing the potential of this treatment regimen for ACTH-secreting pituitary adenomas. This study thus has potentially important implications for novel therapeutic approaches to CD. Copyright © 2014 by the Endocrine Society.

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