Review
The cAMP pathway and the control of adrenocortical development and growth

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Abstract

In the last 10 years, extensive studies showed that the cAMP pathway is deregulated in patients suffering from adrenocortical tumours, and particularly in primary pigmented nodular adrenocortical disease (PPNAD). Here we describe how evidence arising from the analysis of patients’ data, mouse models and in vitro experiments, have shed light on the cAMP pathway as a central player in adrenal physiopathology. We also show how novel data generated from mouse models may point to new targets for potential therapies.

Highlights

► We describe the central role of cAMP pathway deregulations in adrenal pathophysiology. ► We compare mouse models and confront them with data from patients. ► We update mechanisms linking Cushing syndrome to various adrenal tumours. ► We discuss contribution of mouse models to novel understanding of adrenal development.

Introduction

The adult adrenal gland is an encapsulated organ with two major compartments: the medulla, and the cortex. The medulla forms the core of the gland and is essentially composed of neuroendocrine cells, the chromaffin cells that mainly produce catecholamines (adrenaline/noradrenaline). The cortex itself is subdivided in three histologically and functionally different zones. The outer Zona glomerulosa (where cells are organised in glomeruli) (ZG) is the main site for production of aldosterone. Its endocrine activity is controlled by the renin/angiotensin system and is implicated in the long-term regulation of blood pressure. The intermediate Zona fasciculata (where cells are organised in tightly packed cords) (ZF) is responsible for producing glucocorticoids (mainly cortisol in humans) under the control of the adrenocorticotropic hormone (ACTH). Finally, the inner cortical zone, which is in contact with the medulla, is called Zona reticularis (its cells form a kind of net pattern) (ZR). This zone, which is only found in higher primates, produces androgens, mainly DHEA/DHEAS, and develops in the first years of life in humans (reviewed in (Auchus, 2011)).

Whereas chromaffin cells are derived from neuroblasts that migrate to the adrenal primordium during development, all mature cortical cells originate from the foetal cortex within the adrenal (Zubair et al., 2009, Zubair et al., 2008). This foetal cortex also gives rise to the foetal zone, a large transitory zone located in the inner cortex, that essentially produces DHEA/DHEAS during foetal life, and that progressively disappears in post-natal stages. This progressive atrophy is concomitant with the growth of the mature cortex arising from the persistent definitive/transitional zone (Spencer et al., 1999). This post-natal development and later maintenance of the cortex depends on progenitor cells that are located in the subcapsular region of the gland (King et al., 2009).

In mice, adrenal development is roughly comparable with human. However, corticosterone but not cortisol is produced from the Z. fasciculata. This results from absence of Cyp17 expression in adult rodent adrenals (Keeney et al., 1995). Therefore, DHEA cannot be synthesized and no Z. reticularis can be distinguished. The X-zone, which can be considered as the mouse foetal cortical zone, remains present after birth. It disappears through a massive wave of apoptosis during puberty in males, or during the first pregnancy in females (Beuschlein et al., 2003, Holmes and Dickson, 1971). In nulliparous females, the X-zone is maintained for several months, and eventually regresses.

The main role of the cAMP pathway in the adrenal is associated with control of cortisol production. MC2R, the receptor of the pituitary adrenocorticotropic hormone (ACTH), is strongly expressed in ZF cells, and to a lower extent in ZG cells. MC2R is a G protein-coupled receptor. When activated by ACTH, it induces adenylate cyclase activity via Gsα, increasing the intracellular levels of the secondary messenger cAMP. In turn, this rise induces cAMP-dependent protein kinase A (PKA) activation whose targets ultimately stimulate cortisol production and release. PKA is a heterotetramer composed of two catalytic subunits (C) endowed with serine/threonine kinase activity. These are associated with a dimer of regulatory subunits (R) that are the targets of cAMP. Eight genes encode the four C subunits, Cα, β, γ and the most recently characterized Prkx (Zimmermann et al., 1999), and the four R subunits, RIα and β, RIIα and β (Tasken et al., 1997). In the absence of cAMP, the C kinase activity is repressed by interaction with the R subunits. Binding of cAMP molecules to the R subunits induces a conformational change and their dissociation from the PKA tetramer ultimately leading to the release of fully active C subunits (Kim et al., 2007, Kim et al., 2005). Phosphorylated downstream targets of PKA include the CREB protein (cAMP response element-binding), a transcriptional factor inducing transcription of genes whose products are involved in steroidogenesis, such as the steroidogenic acute regulatory protein StAR. Interestingly, StAR, which is responsible for the limiting step of cholesterol transport into the mitochondria, is also directly activated by PKA phosphorylation (Arakane et al., 1997).

Inactivation of PKA catalytic activity follows the termination of adenylate cyclase stimulation. Excess cAMP is degraded by phosphodiesterases (PDE), allowing the PKA inactive tetramer to reform.

Cushing’s syndrome is the result of excessive production and release of cortisol. The two main causes of this excessive cortisol production are ACTH secreting pituitary adenomas, or autonomous activation of the adrenal cortex itself. In the last 10 years, particular emphasis has been put on ACTH-independent Cushing’s syndromes. Autonomous activation of ZF has been linked to induction of the cAMP/PKA pathway resulting from alterations occurring at different levels of signal transduction: ectopic expression of illegitimate G protein coupled receptors, activating mutations of the GNAS gene (encoding Gsα subunit), inactivating mutations of genes encoding RIα (PRKAR1A) or encoding phosphodiesterases (PDE11A4 and PDE8B).

Adrenal overactivity has been associated with tumour formation, raising the question of a possible role of the cAMP pathway in adrenal tumourigenesis. Indeed, presence of ectopically expressed G-protein coupled receptors (Lacroix et al., 2010) or GNAS mutations (Fragoso et al., 2003) were found in ACTH-independent macronodular adrenal hyperplasia (AIMAH). Both PRKAR1A mutations (Bertherat et al., 2009, Kirschner et al., 2000) and PDE mutations (Horvath et al., 2006a, Horvath et al., 2008) have been associated with isolated micronodular adrenocortical disease, notably with PPNAD. Loss of heterozygosity on the wild-type allele was associated with PRKAR1A and PDE11A4 inactivating mutations. Consequently, the corresponding wild-type proteins are absent from the tumours (Groussin et al., 2002, Horvath et al., 2006a). Strikingly, in many of the patients displaying PRKAR1A mutations, PPNAD was detected as part of Carney complex. This pathology is characterised by tumour growth in the skin and heart but also in many endocrine glands in which the tumours induce overactivity. Moreover, altered cAMP signalling, somatic PRKAR1A mutations and somatic losses in PRKAR1A locus have all been reported in sporadic adrenocortical adenomas and carcinomas (Bertherat et al., 2003). These observations suggest that PRKAR1A is a good candidate tumour-suppressor in a number of tissues, including the adrenal.

To try to demonstrate the role of the cAMP pathway members, and particularly PRKAR1A in normal and pathological adrenal growth and development, various animal models have been developed in the last 10 years.

Section snippets

Loss of function of cAMP pathway in the adrenal

Two mouse models are particularly relevant to the study of the role of the PKA pathway in adrenocortical development and maintenance: Mc2r knock-out mice, where no ACTH-Receptor is produced by adrenocortical cells (Chida et al., 2007), and Pomc knock-outs, where no ACTH is produced, but MC2R is present (Coll et al., 2004, Karpac et al., 2005). Both gene inactivation strategies failed to induce adrenal defects during foetal development or the neonatal period but later induced a progressive

Are tumour cells, foetal-like cells with adult markers or adult-like cells with foetal markers?

Recent studies on FAdE, the foetal adrenal enhancer of the Sf1 gene, have shown that all adrenocortical cells, from the subcapsular region to the X-zone, initially arise from cells where the FAdE is active (Zubair et al., 2008). Then, from E14 onwards FAdE activity is maintained in the cells that will give rise to the future X-zone, but is turned off in the precursors of the definitive cortical zone. This suggests that there is not one, but that there are two different populations of

The TGFβ pathway

Many convergent data indicate that TGFβ signalling could be an essential actor in the process of tumour growth in the adrenal. First, Beuschlein et al. demonstrated that activin is able to induce specific apoptosis of X-zone cells (Beuschlein et al., 2003), an observation that correlated with the mechanisms implicated in foetal zone regression in human (Spencer et al., 1999). Activin receptors and Smad2, a critical mediator of the activin/TGFβ intracellular signalling, were strongly expressed

Prkar1a as a tumour suppressor in mice

What was suspected in human, where up to 80% of the PPNAD cases were related to PRKAR1A inactivating mutations (Bertherat et al., 2009), has been confirmed in mice. Mice heterozygous for Prkar1a developed a Carney-like complex, and ablation of RIα in adrenocortical cells induced tumourigenesis. However, mouse models show that tumour development arises from the innermost cortex, and correlates with appearance of foetal markers that were retrospectively found in human samples.

Questions about the origin of the PPNAD tumour in mouse and human

Data from AdKO mice

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