44 research outputs found

    Proliferation of Transformed Somatotroph Cells Related to Low or Absent Expression of Protein Kinase A Regulatory Subunit 1A Protein

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    Abstract The two regulatory subunits (R1 and R2) of protein kinase A (PKA) are differentially expressed in cancer cell lines and exert diverse roles in growth control. Recently, mutations of the PKA regulatory subunit 1A gene (PRKAR1A) have been identified in patients with Carney complex. The aim of this study was to evaluate the expression of the PKA regulatory subunits R1A, R2A, and R2B in a series of 30 pituitary adenomas and the effects of subunit activation on cell proliferation. In these tumors, neither mutation of PRKAR1A nor loss of heterozygosity was identified. By real-time PCR, mRNA of the three subunits was detected in all of the tumors, R1A being the most represented in the majority of samples. By contrast, immunohistochemistry documented low or absent R1A levels in all tumors, whereas R2A and R2B were highly expressed, thus resulting in an unbalanced R1/R2 ratio. The low levels of R1A were, at least in part, due to proteasome-mediated degradation. The effect of the R1/R2 ratio on proliferation was assessed in GH3 cells, which showed a similar unbalanced pattern of R subunits expression, and in growth hormone-secreting adenomas. The R2-selective cAMP analog 8-Cl cAMP and R1A RNA silencing, stimulated cell proliferation and increased Cyclin D1 expression, respectively, in human and rat adenomatous somatotrophs. These data show that a low R1/R2 ratio promoted proliferation of transformed somatotrophs and are consistent with the Carney complex model in which R1A inactivating mutations further unbalance this ratio in favor of R2 subunits. These results suggest that low expression of R1A protein may favor cAMP-dependent proliferation of transformed somatotrophs

    Duplications disrupt chromatin architecture and rewire GPR101-enhancer communication in X-linked acrogigantism

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    X-linked acrogigantism (X-LAG) is the most severe form of pituitary gigantism and is characterized by aggressive growth hormone (GH)-secreting pituitary tumors that occur in early childhood. X-LAG is associated with chromosome Xq26.3 duplications (the X-LAG locus typically includes VGLL1, CD40LG, ARHGEF6, RBMX, and GPR101) that lead to massive pituitary tumoral expression of GPR101, a novel regulator of GH secretion. The mechanism by which the duplications lead to marked pituitary misexpression of GPR101 alone was previously unclear. Using Hi-C and 4C-seq, we characterized the normal chromatin structure at the X-LAG locus. We showed that GPR101 is located within a topologically associating domain (TAD) delineated by a tissue-invariant border that separates it from centromeric genes and regulatory sequences. Next, using 4C-seq with GPR101, RBMX, and VGLL1 viewpoints, we showed that the duplications in multiple X-LAG-affected individuals led to ectopic interactions that crossed the invariant TAD border, indicating the existence of a similar and consistent mechanism of neo-TAD formation in X-LAG. We then identified several pituitary active cis-regulatory elements (CREs) within the neo-TAD and demonstrated in vitro that one of them significantly enhanced reporter gene expression. At the same time, we showed that the GPR101 promoter permits the incorporation of new regulatory information. Our results indicate that X-LAG is a TADopathy of the endocrine system in which Xq26.3 duplications disrupt the local chromatin architecture forming a neo-TAD. Rewiring GPR101-enhancer interaction within the new regulatory unit is likely to cause the high levels of aberrant expression of GPR101 in pituitary tumors caused by X-LAG.The work was supported by the following funding sources: Fondazione Telethon, Italy grant no. GGP20130 (to G.T.); Society for Endocrinology equipment grant (to G.T.); Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD), National Institutes of Health (NIH) Research project Z01-HD008920 (to C.A.S., supporting G.T., F.R.F.); Fonds d’Investissement pour la Recherche Scientifique (FIRS) of the Centre Hospitalier Universitaire de Liège (to A.F.D. and A.B.); the JABBS Foundation, UK (to A.B.); and Novo Nordisk Belgium Educational Grant, Belgium (to A.F.D. and A.B.). M.F. was funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (#800396) and a Juan de la Cierva-Formación fellowship from the Spanish Ministry of Science and Innovation (FJC2018-038233-I). G.T. was funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (#843843). A.F.D. and D.A. were supported by Action de Recherche Concertée (ARC) Grant 17/21-01 from Liège University. D.A. was supported by grants from Télévie (7461117 F, 7454719 F) and the Léon Fredericq Foundation, Belgium

    PAM variants in patients with thyrotrophinomas, cyclical Cushing’s disease and prolactinomas

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    IntroductionGermline loss-of-function variants in PAM, encoding peptidylglycine α-amidating monooxygenase (PAM), were recently discovered to be enriched in conditions of pathological pituitary hypersecretion, specifically: somatotrophinoma, corticotrophinoma, and prolactinoma. PAM is the sole enzyme responsible for C-terminal amidation of peptides, and plays a role in the biosynthesis and regulation of multiple hormones, including proopiomelanocortin (POMC).MethodsWe performed exome sequencing of germline and tumour DNA from 29 individuals with functioning pituitary adenomas (12 prolactinomas, 10 thyrotrophinomas, 7 cyclical Cushing’s disease). An unfiltered analysis was undertaken of all PAM variants with population prevalence <5%.ResultsWe identified five coding, non-synonymous PAM variants of interest amongst seven individuals (six germline, one somatic). The five variants comprised four missense variants and one truncating variant, all heterozygous. Each variant had some evidence of pathogenicity based on population prevalence, conservation scores, in silico predictions and/or prior functional studies. The yield of predicted deleterious PAM variants was thus 7/29 (24%). The variants predominated in individuals with thyrotrophinomas (4/10, 40%) and cyclical Cushing’s disease (2/7, 29%), compared to prolactinomas (1/12, 8%).ConclusionThis is the second study to demonstrate a high yield of suspected loss-of-function, predominantly germline, PAM variants in individuals with pathological pituitary hypersecretion. We have extended the association with corticotrophinoma to include the specific clinical entity of cyclical Cushing’s disease and demonstrated a novel association between PAM variants and thyrotrophinoma. PAM variants might act as risk alleles for pituitary adenoma formation, with a possible genotype-phenotype relationship between truncating variants and altered temporal secretion of cortisol

    Non-functioning pituitary adenoma database: a useful resource to improve the clinical management of pituitary tumors

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    Objective: The long-term outcome of non-functioning pituitary adenoma (NFPA) patients is not clearly established, probably due to the low annual incidence and prolonged natural history of these rare tumors. The aim of this study was to evaluate clinical data at presentation and long-term post-surgery and radiotherapy outcome in a cohort of patients with NFPA. Design and methods: A computerized database was developed using Access 2000 software (Microsoft Corporation, 1999). Retrospective registration of 295 NFPA patients was performed in seven Endocrinological Centers of North West Italy. Data were analyzed by STATA software. Results: The main presenting symptoms were visual defects (67.8%) and headache (41.4%) and the most frequent pituitary deficit was hypogonadism (43.3%), since almost all tumors were macroadenomas (96.5%). Surgery was the first choice treatment (98% of patients) and total debulking was achieved in 35.5%. Radiotherapy was performed as adjuvant therapy after surgery in 41% of patients. At the follow-up, recurrence occurred in 19.2% of patients without post-surgical residual tumor after 7.5 ± 2.6 years, regrowth in 58.4% of patients with post-surgical remnant after 5.3 ± 4.0 years and residue enlargement in 18.4% of patients post-surgically treated with radiotherapy after 8.1 ± 7.3 years. Conclusions: Our database indicates that the goal of a definitive surgical cure has been achieved during the last decade in a low percentage of patients with NFPA. This tumor database may help to reduce the delay between symptom onset and diagnosis, to assess prognostic parameters for the follow-up of patients with different risk of recurrence and to define the efficacy and safety of different treatments and their association with mortality/morbidity

    MANAGEMENT OF ENDOCRINE DISEASE: Bone disorders associated with acromegaly: mechanisms and treatment

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    Growth hormone (GH) and insulin-like growth factor-I (IGF-I) exert physiological actions on the skeleton throughout life, by stimulating longitudinal bone growth in children, the acquisition of bone mass during adolescence and the maintenance of skeletal architecture in adults. When GH and IGF-I are secreted in excess, bone remodeling is enhanced leading to deterioration of bone microstructure and impairment of bone strength. Indeed, acromegaly causes skeletal fragility, and vertebral fractures are reported in a remarkable number of subjects exposed to GH and IGF-I excess. The management of skeletal fragility in acromegaly is a challenge, since the awareness of this complication is low, the prediction of fracture risk is difficult to ascertain, the risk of fractures remains after the control of acromegaly and the effectiveness of bone-active drugs is unknown. This review is an update on bone disorders associated with acromegaly and provides a perspective of possible therapeutic approaches based on emerging pathophysiological and clinical information

    Osteopatia acromegalica: diagnosi e trattamento

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    L’ipersecrezione di GH e IGF-I induce una stimolazione eccessiva del rimodellamento scheletrico con conseguente incremento del riassorbimento osseo, deterioramento della microstruttura scheletrica trabecolare e corticale con aumentato rischio di fratture vertebrali (FV). Circa un terzo dei pazienti affetti da acromegalia sviluppa FV, in stretta relazione con la durata di esposizione a elevati livelli di GH e IGF-I e ad alterazioni microstrutturali sia dell’osso trabecolare che corticale. La gestione dell’osteopatia acromegalica è complessa, in quanto la predizione del rischio di fratture è incerta, il controllo dell’ipersecrezione di GH non sempre favorisce il ripristino della normale struttura scheletrica e la riduzione del rischio fratturativ

    Il ruolo della morfometria vertebrale nella gestione clinica delle complicanze scheletriche della Sindrome di Cushing

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    Le fratture vertebrali sono una complicanza molto frequente della sindrome di Cushing e richiedono una diagnosi radiologica mediante tecnica morfometrica. La morfometria vertebrale è di semplice esecuzione, consiste nel misurare le altezze dei corpi vertebrali e consente di identificare precocemente i pazienti fratturati da sottoporre a terapia osteo-attiva indipendente dal trattamento dell’ipercortisolismo endogeno

    Characterization of Intracellular Signaling Mediated by Human Somatostatin Receptor 5: Role of the DRY Motif and the Third Intracellular Loop

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    Somatostatin (SST) exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1-SST5) linked to multiple cellular effectors. The receptor structural domains involved in these effects have been only partially elucidated. The aim of the study was to investigate the molecular determinants mediating the interaction of the human SST5 with intracellular signaling in the pituitary cell line GH3, focusing on the BBXXB domain in the third intracellular loop and the DRY motif in the second intracellular loop. We analyzed the effects of the SST5 agonist BIM23206 on cAMP accumulation, intracellular calcium, GH secretion, cell proliferation, and ERK1/2 phosphorylation in cells expressing either wild-type SST5 or mutant receptors, in particular the naturally occurring mutant R240W in the BBXXB domain and the D136A and R137A mutants in the DRY motif. We found that residues D136 and R137 were critical for SST5 signaling because their substitutions abolished all the intracellular responses. Conversely, third intracellular loop mutations resulted in receptor that inhibited intracellular cAMP levels similar to the wild-type (50 ± 9 vs. 53 ± 12% inhibition) but failed to mediate the other responses elicited by wild-type SST5, i.e. reduction of intracellular calcium levels as well as inhibition of ERK1/2. These events resulted in an absent inhibition of GH release and an impaired reduction of cell proliferation (38 ± 7 vs. 76 ± 6% inhibition in wild type, P < 0.05). These data indicate that different regions of SST5 are required for the activation of different signaling pathways

    Resistance to dopamine agonists and somatostatin analogues in pituitary tumors: focus on cytoskeleton involvement

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    Pituitary tumors, that origin from excessive proliferation of a specific subtype of pituitary cell, are mostly benign tumors, but may cause significant morbidity in affected patients, including visual and neurologic manifestations from mass-effect, or endocrine syndromes caused by hormone hypersecretion. Dopamine (DA) receptor DRD2 and somatostatin (SS) receptors (SSTRs) represent the main targets of pharmacological treatment of pituitary tumors since they mediate inhibitory effects on both hormone secretion and cell proliferation, and their expression is retained by most of these tumors. Although long acting DA and SS analogs are currently used in the treatment of prolactin (PRL)- and growth hormone (GH)-secreting pituitary tumors, respectively, clinical practice indicates a great variability in the frequency and entity of favourable responses. The molecular basis of the pharmacological resistance are still poorly understood, and several potential molecular mechanisms have been proposed, including defective expression or genetic alterations of DRD2 and SSTRs, or an impaired signal transduction. Recently, a role for cytoskeleton protein filamin A (FLNA) in DRD2 and SSTRs receptors expression and signalling in PRL- and GH- secreting tumors, respectively, has been demonstrated, first revealing a link between FLNA expression and responsiveness of pituitary tumors to pharmacological therapy. This review provides an overview of the known molecular events involved in SS and DA resistance, focusing on the role played by FLNA
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