Date Published: June 10, 2010
Publisher: Public Library of Science
Author(s): Isabelle Sahut-Barnola, Cyrille de Joussineau, Pierre Val, Sarah Lambert-Langlais, Christelle Damon, Anne-Marie Lefrançois-Martinez, Jean-Christophe Pointud, Geoffroy Marceau, Vincent Sapin, Frédérique Tissier, Bruno Ragazzon, Jérôme Bertherat, Lawrence S. Kirschner, Constantine A. Stratakis, Antoine Martinez, G. Stanley McKnight
Abstract: Carney complex (CNC) is an inherited neoplasia syndrome with endocrine overactivity. Its most frequent endocrine manifestation is primary pigmented nodular adrenocortical disease (PPNAD), a bilateral adrenocortical hyperplasia causing pituitary-independent Cushing’s syndrome. Inactivating mutations in PRKAR1A, a gene encoding the type 1 α-regulatory subunit (R1α) of the cAMP–dependent protein kinase (PKA) have been found in 80% of CNC patients with Cushing’s syndrome. To demonstrate the implication of R1α loss in the initiation and development of PPNAD, we generated mice lacking Prkar1a specifically in the adrenal cortex (AdKO). AdKO mice develop pituitary-independent Cushing’s syndrome with increased PKA activity. This leads to autonomous steroidogenic genes expression and deregulated adreno-cortical cells differentiation, increased proliferation and resistance to apoptosis. Unexpectedly, R1α loss results in improper maintenance and centrifugal expansion of cortisol-producing fetal adrenocortical cells with concomitant regression of adult cortex. Our data provide the first in vivo evidence that loss of R1α is sufficient to induce autonomous adrenal hyper-activity and bilateral hyperplasia, both observed in human PPNAD. Furthermore, this model demonstrates that deregulated PKA activity favors the emergence of a new cell population potentially arising from the fetal adrenal, giving new insight into the mechanisms leading to PPNAD.
Partial Text: Primary pigmented nodular adrenocortical disease (PPNAD) is a rare form of bilateral micronodular adrenocortical hyperplasia leading to high morbidity due to ACTH (adreno corticotropic hormone)-independent Cushing’s syndrome. PPNAD may be either sporadic or regarded as the most frequent endocrine manifestation of Carney complex (CNC), an autosomal dominant multiple neoplasia syndrome characterized by cardiac myxomas, spotty skin pigmentation and endocrine overactivity . Cushing’s syndrome in PPNAD is most diagnosed in children and young adults. Both isolated PPNAD and CNC have been associated with inactivating mutations in PRKAR1A, the gene encoding the type 1α regulatory subunit (R1α) of the cAMP-dependent protein kinase (PKA) , . Among CNC patients with Cushing’s syndrome, the frequency of PRKAR1A mutations is about 80%. Tumour-specific loss of heterozygosity within the chromosomal region harboring PRKAR1A is observed in tumours from CNC patients and isolated PPNAD, suggesting that PRKAR1A is a potential tumour suppressor gene . Because general homozygous loss of Prkar1a is lethal in early mouse embryos, various haploinsufficiency and tissue-specific knock-out models have been engineered to demonstrate its tumour suppressor activity , . General down-regulation of R1α levels has been achieved either in mouse lines heterozygous for a null allele of Prkar1a,  or in a transgenic line carrying an inducible antisense-construct . Both approaches indicate that haploinsufficiency for Prkar1a predisposes to tumour formation in a spectrum of endocrine and non-endocrine tissues that are cAMP-responsive; the mouse phenotype partially overlaps with the human one. However haploinsufficiency in mouse models does not appear to be sufficient to promote tumour formation in a subset of tissues known for their propensity to develop neoplasms in CNC patients. Thus, complete loss of Prkar1a using heart-, Schwann cell- or pituitary-specific knockouts was required to induce tumours in these tissues –. To date, although PPNAD is the most frequent endocrine disorder observed in CNC patients , little is known on its pathophysiology. No clear adrenal lesions nor Cushing’s syndrome were observed in mouse models of haploinsufficiency, suggesting that complete loss of Prkar1a might be required to phenocopy human phenotype. To address directly this question and obtain a possible mouse model for PPNAD, we produced mice with targeted Prkar1a gene inactivation in adreno-cortical cells by mating Prkar1a floxed mice with Akr1b7-Cre mouse line, a Cre expressing line allowing specific gene ablation in the steroidogenic lineage of the adrenals . Adrenal cortex-specific Prkar1a knockout mice (AdKO) develop pituitary-independent Cushing’s syndrome and evident signs of deregulated adreno-cortical cells differentiation and hyperplasia. These defects lead to improper maintenance and expansion of foetal adrenal cells in adult adrenals and establishment of tumoural conditions. Deregulation of the inhibin-activin signalling pathway seems to be implicated in this improper maintenance in AdKO mice model and in the human pathology. Our data provide the first in vivo evidence that the absence of R1α subunit of PKA is sufficient to induce the autonomous adrenal hyper-activity and bilateral hyperplasia observed in PPNAD. They also strongly suggest that deregulated PKA activity positively affects the maintenance of foetal characteristics in adult adrenal glands.
Here, we shown that the adrenal-specific ablation of Prkar1a, the Carney Complex gene 1 (CNC1), in mouse reproduced the essential features of PPNAD observed in humans carrying PRKAR1A mutations. AdKO mice developed ACTH-independent Cushing’s syndrome and cortical hyperplasia combined with atrophic areas that are typical hallmarks of PPNAD . This mouse model definitively proves the central role of PRKAR1A gene defects in the etiology of PPNAD. Furthermore, the discovery of an unexpected role of Prkar1a in the repression of foetal features in adrenal cortex provides novel mechanistic insight into the cellular dynamics leading to definitive adrenal tissue or, when disturbed, to morbid hyperplasia.