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. Author manuscript; available in PMC: 2013 Jun 11.
Published in final edited form as: Mol Cell Endocrinol. 2011 Oct 15;351(1):28–36. doi: 10.1016/j.mce.2011.10.006

The cAMP pathway and the control of adrenocortical development and growth

Cyrille de Joussineau a, Isabelle Sahut-Barnola a, Isaac Levy b, Emmanouil Saloustros b, Pierre Val a, Constantine A Stratakis b, Antoine Martinez a,*
PMCID: PMC3678347  NIHMSID: NIHMS460456  PMID: 22019902

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.

Keywords: cAMP, PKA, Adrenal, Pathology

1. Introduction

1.1. Histological and functional introduction to the adrenal gland

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, 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.

1.2. Role and description of the cAMP pathway in adrenocortical function

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, 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.

1.3. cAMP pathway deregulation and its link to human adrenal pathologies

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.

2. Impact of cAMP pathway activity on the adrenal: from little to plenty of activity

2.1. 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 atrophy of the ZF. Interestingly, ACTH supplementation of POMC-null mice restored adrenal size, cortex morphology and glucocorticoid secretion through a hypertrophic response of the ZF cells (Coll et al., 2004). In contrast, transplantation of Pomc−/− adrenals in wild-type animals restored the hyperplastic response (Karpac et al., 2005). These data suggest that POMC peptides are dispensable for foetal adrenal development but are required for postnatal proliferation and maintenance of adrenal structures. Importantly, atrophy of MC2R-null adrenals was not accounted for by a decrease in the number of ZF cells (Chida et al., 2007). This could indicate that in addition to its innocuity on adrenal development, absence of ACTH-dependent activation of the cAMP pathway does not impair the establishment of a normal stock of progenitor cells. Thus, the progressive ZF atrophy could be interpreted either as a differentiation defect, where progenitors would not be recruited in the absence of cAMP signalling, or as a possible increase in ZF cells death rate that would not be compensated for by a basal recruitment of progenitors. Of note, as in the first hypothesis, the progenitors are supposed to be subcapsular cells that do not express the ACTH receptor. The observed defect should thus result from an indirect mechanism.

These mouse phenotypes reflect what is seen in human with Familial Glucocorticoid deficiency type 1, a disease mostly resulting from mutations in MC2R that impair its trafficking to the membrane (Chung et al., 2008). This shows that, overall, lack of cAMP pathway activity in adrenal does not induce developmental defects, but that this activity is required postnatally, to produce cortisol and to maintain the structure of the ZF.

2.2. Gain-of-function of the cAMP pathway in adrenal cells

At the other end, different models were designed to reproduce hyperactivity of the cAMP pathway in the adrenal.

One strategy was to recapitulate ectopic expression of G protein-coupled receptors (GPCRs) found in some AIMAH using xenotransplantation models of bovine adrenocortical cells, transduced with viral expression vectors (Mazzuco et al., 2006a,b). These xenografted mice developed benign tumours, indicating that ectopic expression of a single GPCR was sufficient to induce phenotypic changes leading to Cushing’s syndrome and tumour transformation. However, mechanisms leading to tumour formation are difficult to assess in transplantation models and thus participation of cAMP-dependent vs-independent pathways in cell transformation mediated by GPCR remains to be established.

Another strategy was to decrease the degradation of cAMP by reducing phosphodiesterase activity. Both PDE8B and PDE11A4 have been found mutated in some patients developing adrenal hyperplasia, in association with loss of heterozygosity at the chromosomal region containing the PDE11A4 gene (Horvath et al., 2006a). In Pde8b KO mice, mild hypercorticosteronemia was detected in adults, but no tumour growth was shown. This could suggest that increased PKA activity is not sufficient to promote developmental defects or tumour growth, even though it is sufficient to induce a Cushing’s syndrome. It is consistent with the human data where PDE11A mutations were considered as phenotype modifiers of already pre-existing conditions resulting from PRKAR1A mutations (especially as an enhancer of phenotype in males) (Libe et al., 2011). Similarly, PDE8B variants seemed to predispose to adrenal defects, but were not clearly shown to promote adrenal tumour formation (Horvath et al., 2006a, 2008).

A third strategy targeted Prkar1a, the most promising tumour-suppressor candidate of the pathway. Total Prkar1a KO mice have severe heart development defects causing their death at E9.5 (Amieux et al., 2002). Haplo-insufficiency of Prkar1a induces tumour growth in thyroid, Schwann cells, bone, and liver but not in the adrenal. These observations showed that Prkar1a was a tumour-suppressor gene in many tissues, but evidence was lacking for its specific role in the adrenal (Kirschner et al., 2005; Veugelers et al., 2004). A more severe loss of RIα protein was achieved by an antisense strategy, which resulted in decreases up to 70% in some tissues (Griffin et al., 2004). Although the decrease in RIα protein in the adrenal did not reach 50%, half of the transgenic mice presented with adrenal hyperplasia. Interestingly, histological defects suggesting that foetal X-zone was maintained were observed. This indicated that some of the phenotypes could result from developmental alterations.

In order to overcome the previous technological limitations, we generated an adrenocortical-specific KO of Prkar1a (AdKO) (Sahut-Barnola et al., 2010). In our system, genetic ablation occurs in adrenocortical cells at E14.5 and targets all cortical subtypes in adults (Lambert-Langlais et al., 2009). This targeted KO induced the appearance of a PPNAD-like syndrome in mice, with the development of an age- and sex-dependent Cushing’s syndrome (males adrenals were much less affected by the KO, which correlates with the human pathology that develops more frequently in women (Bertherat et al., 2009)), and subsequent tumour formation. Interestingly, this was correlated with the growth of a population of large cells at the medullocortical boundary that expressed both X-zone (20αHSD) (Hershkovitz et al., 2007) and ZF (Akr1b7) specific markers (Aigueperse et al., 2001). Centrifugal extension of this foetal-like zone coincided with atrophy of the normal ZF. A possible hypothesis for this ZF atrophy could be that continuous and autonomous production of corticosterone repressed the production of ACTH by a regulatory feedback loop. Interestingly, plasma ACTH levels were mildly decreased in this mouse model even though were not blunted (Sahut-Barnola et al., 2010). Therefore, autonomous corticosterone production could result in a “Pomc KO-like” or “Mc2r KO-like” phenotype in the ZF, indicating that in AdKO mice, cells of the “normal” ZF and/or progenitor recruitment could still be partially dependent on ACTH release by the pituitary. To elucidate the aetiology of the phenomenon resulting in ZF atrophy, expression of progenitor markers was quantified in AdKO mice. These were not altered (Sahut-Barnola et al., 2010), which was reminiscent of Pomc/Mc2r KO mice. We also tested potential decay of ZF cells in AdKO mice. In AdKO mice, adrenocortical cells displayed increased resistance to apoptosis (Sahut-Barnola et al., 2010). However, we detected high β-galactosidase activity in the inner ZF cells lining the hypertrophic cells of the foetal-like zone, a phenomenon that is typically associated with senescence (Fig. 1). Hence, ZF atrophy may result from abnormally fast ageing of cells in the adrenal-specific Prkar1a KO mouse model.

Fig. 1.

Fig. 1

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Senescence-associated β-galactosidase staining in adrenals from 14-month-old females in WT and AdKO mice. C, cortex; XL, X-like zone; M, medulla; HE, βbars, 50 µm.

Interestingly, AdKO mouse model is globally reminiscent of the situation in PPNAD patients’ tissues. In that case, the nodules that form the tumours completely lack PRKAR1A following loss of heterozygosity. They are also made of very large cells surrounded by atrophic internodular ZF tissue, these combined defects resulting in a hyperplastic but normal-sized adrenal. Similar to AdKO cells, they express high levels of the foetal marker inhibinα, suggesting that a developmental defect could also be involved in the establishment of the pathology in human.

3. Origin of the tumour cells in the mouse adrenal cortex lacking RIα

3.1. 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 progenitors, one for the X-zone, and one for the definitive adult zone.

Because of their position and the markers that they express, foetal-like cells emerging from the innermost cortex of AdKO adrenals seem to be derived not from adult progenitors, but from foetal cells. This would imply that first, there is actually a population of foetal progenitors that lies at the medulla/cortex boundary, and second, that for some reason, this population is stimulated and proliferates to produce the tumour expansion that is seen in the AdKO adrenal. Several arguments are in favour of this model.

First, in adult male mice, castration induces the appearance of a secondary X-zone (Hirokawa and Ishikawa, 1975). Given that primary X-zone cells segregate from adult cortical cells early in development (Zubair et al., 2006), it seems logical to explain this phenomenon by reactivation of a dormant progenitor population lying in the innermost cortex. Second, normal ZF cells in AdKO model seem to enter senescence (Fig. 1). It is thus unlikely that they are later recruited as precursors of the tumour cells. Third, in mice where activating mutation of β-catenin is targeted in the adrenal cortex (ΔCat mice), tumour growth first occurs at the juxtamedullary region and results in invasion of the medulla. Only later on does the tumour develop from the subcapsular region where adult cortical progenitors are found (Berthon et al., 2010). This would indicate that there really is a population of dormant precursors that lie in the inner cortex of the adult adrenal (Fig. 2).

Fig. 2.

Fig. 2

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Dysregulation of PKA-Wnt signalling pathways influences zonal identities and promotes tumours. Loss of RIα in adrenocortical cells (AdKO) leads to the resurgence, the expansion and the centrifugal differentiation of foetal-like cells that acquire fasciculata markers, concomitantly to an atrophy of fasciculata cells in the adult cortex. AdKO mice develop morbid hyperplasia causing ACTH-independent Cushing syndrome reminiscent of PPNAD in human (Sahut-Barnola et al., 2010). Constitutive activation of β-catenin in adrenocortical cells (ΔCat) leads to expansion of tumours that originate from both the outer cortex and the medullo-cortical boundary. There, tumourigenic cells expand centripetally and differentiate into ectopic glomerulosa to develop into Conn adenomas, at the expense of both fasciculata cells and medulla. Eventually, these benign adenomas may progress into carcinomas (Berthon et al., 2010). P, adult cortex progenitors; P?, hypothetical foetal cortex progenitors; Ca, capsule; G, Zona glomerulosa; F, Zona fasciculata; X, X-zone

3.2. How could foetal-like cells display tumour potential in AdKO mice?

Two non-mutually exclusive mechanisms can account for recruitment of foetal-like cells for tumour formation: repression of an inhibitory mechanism, which prevents their recruitment in a normal setting, or activation of their recruitment. In AdKO mice, adrenocortical cells displayed increased resistance to apoptosis, indicating that a phenomenon of protection against death signals was likely to be involved in tumour growth (Sahut-Barnola et al., 2010). Interestingly, apoptosis is the normal mechanism of clearance of cells in the X-zone (Beuschlein et al., 2003). Resistance to apoptotic signals could favour the growth of foetal-like cells in an environment that would normally be hostile for them. Resistance could be the direct result of ectopic activation of the cAMP pathway in X-zone cells. Indeed, normal X-zone cells being devoid of MC2R, they are insensitive to ACTH, the main activator of cAMP pathway in adrenal cortex. However, other pathways are likely to be involved in the tumour process.

4. Molecular mechanisms of adrenal tumour formation in mice lacking RIα

4.1. 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 in the X-zone. In a context of high incidence of adrenal tumours arising from the inner zone of the cortex, actual development of tumours negatively correlated with levels of Smad2. Interestingly, down-regulation of PRKAR1A in H295 cells that are derived from adrenocortical cancer induced resistance to apoptosis. This has been attributed to a severe decrease in SMAD3 protein levels, SMAD3 having the same role as SMAD2 in the TGFβ signalling pathway (Ragazzon et al., 2009). Finally, in AdKO mice, overexpression of inhibinα and follistatin, both inhibitory ligands of activin, was found, and correlatively, inhibinα was detected at high levels in the cells forming the tumours in PPNAD patients’ tissues (Sahut-Barnola et al., 2010). Taken together, these data show that down-regulation of the gene coding the RIα subunit of PKA decreases the activity of a pathway which is implicated in adrenal foetal zone clearance.

However, TGFβ pathway downregulation could be related to the inhibition of a repressive system rather than to real promotion of tumourigenesis. At least two other pathways known to have strong relevance in tumour growth and in cancer were also found altered in PPNAD tissues.

4.2. The Wnt/β-catenin pathway

The Wnt/β-catenin signalling pathway is widely implicated in the control of cell proliferation and differentiation, deregulation of this pathway being found in many cancers. Targeting the early loss of Ctnnb1 gene (encoding β-catenin) in developing adrenocortical cells induces complete failure of adrenal at birth, while lowering the number of cells with β-catenin inactivation only affects cortical cell renewal in adults (Kim et al., 2008). This nicely shows that, as opposed to cAMP/PKA pathway, Wnt/β-catenin pathway is essential to both adrenal development and growth. Activating mutations of the CTNNB1 gene were frequently detected in both adrenocortical adenomas and carcinomas (Bonnet et al., 2011; Gaujoux et al., 2011; Tissier et al., 2005). This strongly argues for an oncogenic role of β-catenin in the adrenal cortex. This was definitively proven by the ΔCat mouse model, where adrenocortical specific expression of a constitutively active β-catenin induced hyperplasia and carcinogenesis in late stages. Interestingly, CTNNB1 gene somatic mutations have been found in adrenal tumours developing secondarily in patients with PPNAD, as well as in patients suffering from Carney complex (Gaujoux et al., 2008; Tadjine et al., 2008). This could suggest that in rare cases, cAMP pathway overactivation by favouring cell survival could also favour appearance of secondary genetic hits targeting β-catenin in order to promote growth of a more aggressive tumour. A more frequent related event is the overexpression of genes related to the Wnt/β-catenin signalling pathway such as WNT1-inducible signalling pathway protein 2 (WISP2), GSK3β and CTNNB1. These were detected in PPNAD tissues and other adrenal hyperplasia related to excessive cAMP signalling (Bourdeau et al., 2004; Horvath et al., 2006b). This regulation defect may depend on some microRNAs (Bimpaki et al., 2010; Iliopoulos et al., 2009). Overall, increased activity of cAMP/PKA signalling pathway is at the origin of adrenocortical tumour growth, and its positive effect on Wnt/β-catenin signalling could either be the founder step of tumour growth or a way to reinforce an already existing tumour potential of adrenocortical cells.

4.3. The mTOR signalling pathway

In two papers, Mavrakis et al., showed that mTOR is activated in cells of the nodules that lack PRKAR1A in PPNAD tissues. In this context, RIα seems to behave as a regulatory subunit for mTOR (Mavrakis et al., 2006, 2007). Deregulation of mTOR pathway is found in many tumours (Grozinsky-Glasberg and Shimon, 2010; Zoncu et al., 2011). Therefore, increased mTOR activity through loss of R1α could support adrenocortical tumour formation (Doghman et al., 2010). In this model, R1α dependent deregulation of mTOR would be completely independent of catalytic PKA activity. The possibility that RIα could exert activities independently of PKA is intriguing. This could account for the more penetrant tumour promoting activity of PRKAR1A mutations compared with PDE mutations that would preferentially impact PKA catalytic activity (Section 2.2.). Interestingly, as in PPNAD, tumour cells in AdKO mice were hypertrophic, which is a hallmark of mTOR activation. Further analysis of the role of mTOR pathway in adrenocortical tumourigenesis is in progress in AdKO mouse model.

5. From mouse models back to human development/pathology

5.1. 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.

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

Data from AdKO mice strongly suggest the existence of a population of dormant adrenocortical progenitors in the juxtamedullary part of the cortex. The fact that in tumours from ΔCat mice (Berthon et al., 2010) the same apparent mobilisation of inner progenitors occurs upon β-catenin activation strongly reinforces this hypothesis (Section 3.1.). More interestingly, these potential progenitors could differentiate into ectopic ZG under the influence of Wnt/β-catenin signalling (ΔCat model), or pseudo ZF (foetal-like zone with ZF markers) under RIalpha ablation-dependent increased cAMP/PKA signalling (AdKO model). This indicates that these progenitors have plasticity in their possible fate (Fig. 2).

In human, ZR arises in the inner cortex at 5–7 years of age, in a process called the adrenarche (Havelock et al., 2004). ZR thus appears after regression of the foetal zone, but interestingly shares the same DHEA/DHEAS steroid production activity. Previous data indicated that ZR probably arises from transdifferentiation of ZF cells. The localisation of the ectopic zone in AdKO mice suggested that it could represent a pseudo-ZR. ZR is characterised by DHEA/DHEAS production that requires the presence of CYP17, but is also favoured by low 3βHSD expression (3βHSD competes for pregnenolone, the substrate CYP17 metabolizes into 17-hydroxypregnenolone) and high CYTB5 levels (that facilitates the 17–20 lyase activity of CYP17 to transform 17-hydroxypregnenolone into DHEA) (Dharia et al., 2004; Mapes et al., 1999; Narasaka et al., 2001; Nguyen et al., 2009). We did not detect circulating or intra-adrenal DHEA/DHEAS in AdKO mice (Sahut-Barnola personnal communication). However, Cyp17 was expressed and functional in the foetal-like cells, which were indeed able to produce cortisol (Sahut-Barnola et al., 2010). Altogether these data also suggest that mouse Cyp17 may have low lyase activity. Moreover, both low 3βHSD and high Cytb5 levels were expressed in AdKO mice when compared to WT (Fig. 3). This situation is reminiscent of both human foetal zone and human adult ZR (Dharia et al., 2004; Narasaka et al., 2001). Furthermore, these findings in mice have to be compared to recent data obtained from the study of marmoset. In this primate, no ZR can be detected in males on the basis of histological and functional criteria (Pattison et al., 2005). However, ZR as revealed by Cytb5 expression and adrenal DHEA production can be detected in adult females. Surprisingly, appearance of this ZR depends on social status: dominant ovulatory females show a faint Cytb5 staining, which is increased in anovulatory subordinate females. Ovariectomized marmosets display an even stronger and expanded staining (Pattison et al., 2007). This suggests that there is a population of “ZR progenitors” that can be mobilized in adult marmosets, depending on gender and ovulatory status. Alternatively, this expansion could also occur by a transdifferentiation of ZF cells. The mouse model could simply differ from primates or it could suggest alternative possibilities that should be considered in future studies.

Fig. 3.

Fig. 3

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Cytb5 and 3βHSD expression in WT and AdKO adrenals. A–B, Immunodetection of 3βHSD (A) and Cytb5 (B) in female adrenals. In 18-month-old AdKO mice, full expansion of foetal-like cortex (low 3βHSD staining) is revealed. Remainings of the atrophic adult cortex, displaying higher 3βHSD staining, is delineated by dotted lines. C, Quantitative representation (RT-qPCR) of Cytb5 mRNA levels in adrenals of 12-month-old females. D, Levels of Cytb5 and 3βHSD were quantified by western blotting in adrenals of 12-month-old females. **P < 0,01. C, cortex; XL, X-like zone; M, medulla; HE, haematoxylin and eosin; Scale bars, 50 µm.

5.3. How to improve mice models? A matter of microenvironment?

Mice models with strict Prkar1a haploinsufficiency were unable to develop ACTH-independent Cushing’s syndrome. The antisense strategy resulting in a loss of 50% of RIα protein in adrenal cells was able to promote defects in the inner cortex of the adrenal (Section 2.2.). Total Prkar1a knockout in the population of adrenocortical steroidogenic cells (AdKO) induced clear hyperplasia, foetal characters resurgence, gender predilection and adult cortex atrophy. However, in addition to all of these characters, other criteria found in PPNAD in human were absent in mouse models. Indeed, hallmarks of Wnt/β-catenin pathway activation were frequent in PPNAD (Gaujoux et al., 2008; Tadjine et al., 2008), but were never observed in any of the mouse models. Another evident criterion is the nodular aspect of the dysplasia in PPNAD patients, which contrasts with the emergence of a new adrenocortical zone in mouse models. Also, the neuroendocrine differentiation of the cells composing the nodules in human (Stratakis et al., 1999) was never detected in mice. Finally, we could not show paradoxical response to dexamethasone in AdKO mice, even though this is one of the diagnostic factors in patients (Stratakis et al., 2001). Indeed, although dexamethasone should result in repression of the HPA axis and subsequent reduction in plasmatic cortisol levels, PPNAD patients respond to this treatment by increased cortisol production. This paradoxical response is attributed to overexpression and abnormal PKA-coupling of the Glucocorticoid Receptor (GR) within the nodules (Bourdeau et al., 2003; Louiset et al., 2009). These four defects were not found in any of the mouse models where RIα levels were altered in the adrenal. However in contrast with patients, most mouse models had decreased Prkar1a expression but not complete loss of RIα in all the cell-types that compose the adrenal. On the contrary, the AdKO model exhibited complete loss of Prkar1a but this was restricted to the steroidogenic cells of the cortex. In the only model in which some loss of heterozygosity (and consequently complete knockout of Prkar1a) was detected in some adrenal cells, nodular organisation was observed within the hyperplastic area (Griffin et al., 2004). One hypothesis that could account for these observations is that the loss of Prkar1a in steroidogenic cells is directly responsible for all the phenotypes that were described in the AdKO model. Under this hypothesis, other phenomena that were detected in PPNAD, i.e. the nodular shape of the tumours, neuroendocrine differentiation, ectopic GR expression and paradoxical response to dexamethasone would be dependent on Prkar1a haploinsufficiency in the other cell-types residing within the adrenal. To address this question, we are currently analysing mice in which genetic ablation of RIα in steroidogenic cells is combined with haploinsufficiency in all other cell types within the adrenal. This should recreate an accurate picture of the microenvironment found in the context of the human pathology.

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