C H A P T E R

13AAnimal Models of Adrenal Genetic Disorders

Felix BeuschleinEndocrine Research Unit, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany

INTRODUCTION

Mouse models exhibit a wide range of possibilities for the investigation of adrenocortical function and growth. Incidental discovery of adrenal tumors or disorders in steroidogenesis in genetically modified animals can provide clues on pathways involved in adrenal func-tion that would not have been predicted on the basis of structural analysis or in vitro exploration. Mouse mod-els can also be used to verify functional significance of a given candidate gene for adrenal growth and steroido-genesis in vivo through targeted genetic modification. Furthermore, incidence of adrenal disorders in inbred mouse strains can serve as the starting point for genetic approaches to identify the underlying genetic cause. In contrast, differences in phenotypic alterations in humans and mice following the same genetic cause can provide important insights into genetic or environmental modi-fiers that impact disease severity. In this chapter we will provide an overview on adrenocortical disease models relevant for either mechanistic studies or future thera-peutic approaches.

MOUSE MODELS FOR CONGENITAL ADRENAL HYPOPLASIA

Cells within the coelomic epithelium give rise to the adrenogonadal primordium, which resembles the primi-tive organs for the adrenals and gonads. Proper adrenal development is dependent upon a cascade of molecular events that includes sequential gene expression, pres-ence of signaling molecules, and paracrine and endo-crine factors [1]. Accordingly, defects in either of these mechanisms can result in adrenal agenesis or aplasia [2]. In fact, a number of mouse models have been described in which absence of the adrenal gland has been part of

Genetic Steroid Disorders. http://dx.doi.org/10.1016/B978-0-12-416006-4.00026-0 3

their phenotype. These alterations mostly include tran-scription factors that are involved in the developmental program of the urogenital ridge as the common precur-sor of the adrenal cortex, the gonads, and the kidney, or, more specifically, that of the adrenogonadal primordium or the adrenal gland itself. Examples include Wilms’ tumor gene 1 (Wt1) [3], Pbx1 [4,5], steroidogenic factor 1 (Sf1) [6], and Cited2 [7]. However, in many instances (including WT1, PBX1 and CITED2) an adrenal phe-notype in affected human patients has been absent or has not been reported. Conversely, in the case of DAX1 affected patients typically suffer from adrenal insuffi-ciency because of cytomegalic adrenal hypoplasia [8,9], while adrenal function is normal in mice with targeted deletions of Dax1 [10].

Despite these differences there are clear examples for the convergence of genotype/phenotype correla-tion among patients and the according mouse models (Table 13A.1). One of the best studied models includes that of mice with targeted deletions of Sf1 [6]. In the mouse embryos, the adrenogonadal primordium starts to express SF1 at approximately 9.0 days post coitum [11]. Thereby, SF1 marks the steroidogenic precursors, which later become adrenal cortex and somatic cells in the gonads. Mice deficient in Sf1 die soon after birth as a result of primary adrenal insufficiency owing to complete absence of adrenal glands – in addition to loss of gonads, pituitary gonadotropes, and ventrome-dial hypothalamus. The essential role of Sf1 for adrenal growth has also been underscored by studies in haplo-insufficient animals, which display a defect in specific adrenal growth paradigms [12] as well as in transgenic models where increase in Sf1 dosage has been shown to induce adrenal tumorigenesis [13]. Interestingly, high expression levels of SF1 can also be regarded as a prognostic marker in human adrenal carcinogenesis [14]. Taken together, these studies have placed SF1 as

Copyright © 2014 Elsevier Inc. All rights reserved.23

13A. ANIMAL MODELS OF ADRENAL GENETIC DISORDERS324

TABLE 13A.1 Examples of Single Gene Defects in Inherited Forms of Primary Adrenal Insufficiency and/or Congenital Adrenal Hyperplasia

Disease OMIM Gene Mouse model Reference

Congenital adrenal hypoplasia 300200300473

NR0B1 (DAX1)NR5A1 (SF1)

KnockoutKnockout

[10] #

[6]

Familial glucocorticoid deficiency– Type 1– Type 2– Type 3– Triple A syndrome

202200609196609197202110

MC2RMRAPunknown/ NNTAAAS

Knockout–Genetic VariantKnockout

[20][19][26]

Congenital adrenal hyperplasia– 21-Hydroxylase deficiency– 11β-Hydroxylase deficiency– 3β-Hydroxysteroid dehydrogenase deficiency– 17α-Hydroxylase deficiency– Lipoid adrenal hyperplasia

201910202010109715202110201710118485

Cyp21Cyp11B1HSD3B2Cyp17STAR, Cyp11a1

Genetic variantKnockout––KnockoutKnockout

[31][38][40][49]

X-linked adrenoleucodystrophy 300371 ABCD1 Knockout [59] *

Autoimmune polyglandular syndrome type 1 240300 AIRE Knockout [60] #

* No reported adrenal phenotype;# no functional relevant adrenal phenotype.(Adapted from ref. [18])

the crucial determinant of adrenocortical growth and differentiation.

MOUSE MODELS FOR FAMILIAL GLUCOCORTICOID DEFICIENCY (ACTH

RESISTANCE SYNDROMES)

Familial glucocorticoid deficiency (FGD) [Online Mendelian Inheritance in Man (OMIM) #202200] is an autosomal recessive disorder resulting from resistance to the action of adrenocorticotropic hormone (ACTH) on the adrenal cortex [15]. Affected individuals present with signs of cortisol deficit such as severe hypoglycemia or severe infection in infancy or childhood. In contrast to other forms of primary adrenal insufficiency, mineralo-corticoid secretion is typically not compromised.

Mutations of the ACTH receptor (melanocortin 2 receptor, MC2R) are responsible for 25% of FGD cases (FGD type 1) [16], while mutations of the MC2R acces-sory protein MRAP, which plays a role in the traffick-ing of MC2R from the endoplasmic reticulum to the cell surface, account for around 15% of FGD cases (FGD type 2) [17,18]. Only recently, mutations in NNT (encoding nicotinamide nucleotide transhydrogenase) have been added to the list of genes causing FGD [19].

In 2007 mice with targeted deletions of the Mc2r were described which resemble most of the features of patients with FGD type 1 [20]. Interestingly, while most knockout animals die from lung failure, depending on the back-ground strain, some Mc2r-deficient animals survive until adulthood even without glucocorticoid substitution [21].

In addition to its steroidogenic-inducing properties, ACTH has been implicated to be required for proper adrenal development and growth [22]. Therefore, it is not surprising that Mc2r knockout mice are affected with significant adrenocortical hypoplasia in comparison to wild-type controls [20]. Ultrastructural investigation of cells from the zona fasciculata in Mc2r knockout mice revealed a diminished number of lipid droplets as well as structural changes in mitochondrial appearance com-pared with that of wild-type animals. In contrast to the clinical phenotype of FGD type 1 patients but similar to that of mice deficient of POMC [22], Mc2r knockout mice were found to secrete aldosterone at reduced levels.

These findings are indicative of a more significant role of ACTH in zona glomerulosa development and func-tion in rodents than is the case in the human. However, there are examples of FGD patients with a homozygous nonsense mutation of the MC2R with a slight degree of mineralocorticoid deficiency as indicated by a raised renin level [23]. Therefore, it is possible that the pheno-type in Mc2r null animals represents a disease severity that is not necessarily found in patients with some resid-ual MC2R activity.

While no mouse model dedicated to FGD type 2 has yet been introduced in the literature, animals with NNT mutations have been described in the context of NNT’s newly established role in adrenal steroidogenesis [19]. NNT represents a highly conserved gene encoding an integral protein of the inner mitochondrial membrane involved in NADPH production. Upon identification of NNT mutations in families affected by FGD, the adre-nal phenotype of mice carrying a natural occurring NNT

MouSE MoDElS FoR ConGEnI

mutation was characterized [19]. Thereby, mutant mice were found to have lower basal and stimulated levels of corticosterone than their wild-type counterparts. While adrenal cortices of affected animals displayed a slightly disorganized zona fasciculata with higher levels of apoptosis, no observable differences in the levels of the steroidogenic enzymes CYP11A1 and CYP11B1 were evident. Overall, these findings suggest that defects in oxidative stress response can result in impaired adreno-cortical function.

In line with this pathophysiological mechanism, adrenal insufficiency in the context of triple A syndrome (also known as Allgrove syndrome; OMIM #231550) has been associated with an impaired oxidative stress response. The triple A syndrome is a rare autosomal recessive disorder characterized by the clinical triad of achalasia of the cardia, alacrima, and ACTH-resistant adrenal insufficiency. The predicted product of AAAS, ALADIN (for alacrima–achalasia–adrenal insufficiency neurologic disorder), belongs to the WD-repeat fam-ily of regulatory proteins [24]. Defects in this nuclear pore protein result in impaired nuclear import of DNA repair and antioxidant proteins, thereby rendering the cells more susceptible to oxidative stress [25]. Mice lacking the nuclear pore complex protein ALADIN show female infertility but fail to develop a phenotype resembling human triple A syndrome [26]. Therefore, the wide range of disease severity, the obvious lack of a genotype/phenotype relationship in human patients, and the absence of a drastic phenotype in Aaas knock-out animals suggest that additional factors, such as environmental influences or modifier genes, contribute to the disease course to a greater extent than previously anticipated.

MOUSE MODELS FOR CONGENITAL ADRENAL HYPERPLASIA

In contrast to humans, the adrenal steroid biosynthe-sis in rodents is characterized by a lack of 17-hydroxy-lase activity [27]. Therefore, pregnenolone is catalyzed by 21-hydroxylase (Cyp21) to corticosterone as the major glucocorticoid, and corticosterone is further hydroxyl-ated to aldosterone, which is the main active mineralo-corticoid in the mouse [28]. Owing to the lack of adrenal 17-hydroxylase activity within the mouse adrenals, ste-roid precursors do not, however, shunt into the andro-gen pathway and no clinical effects (such as virilization of the external genitalia) can be expected in animals that lack upstream steroidogenic enzyme activity. Despite these well-known differences for a number of steroido-genic defects specific mouse models for congenital adrenal hyperplasia (CAH) have been generated. Their phenotypic work-up has provided a number of insights

TAl ADREnAl HyPERPlASIA 325

into genetics and/or functional characteristics of the specific diseases.

21-Hydroxylase Deficiency (21-OHD)

The gene coding for human CYP21 (CYP21A2) is located in the major histocompatibility complex (MHC) on chromosome 6p21.3 while a non-functional pseu-dogene (CYP21A1-P) is located in close proximity [29]. While both genes show a 98% sequence similarity, CYP21A1-P carries several inactivating mutations. In humans, mutations causing 21-OHD are most frequently the result of complex recombination events between CYP21A2 and CYP21A1-P. Interestingly, the human and murine 21-hydroxylase gene locus shows a high level of homology: in both species within the class III region of the MHC two structurally homologous genes for 21-hydroxylase are located in the same region [30].

In 1987 a deleterious phenotype in mice that had undergone meiotic recombination in parts of the H-2 class III region was described, and recombination between homologous chromosomes of the H-2a and H-2wm7 haplotypes causing the deletion of complement compo-nent C4 and the 21-hydroxylase gene was assumed [31]. Following extensive genetic analysis of the Cyp21 locus, it was demonstrated that the underlying molecular defect of 21-OHD in the congenic H-2aw18 mouse strain in fact consists of a complex gene rearrangement caused by unequal crossing over, which generates a hybrid gene consisting of a truncated active gene and pseudo-gene [32]. Multiple missense mutations and a nonsense point mutation resulting in a premature stop codon were found to be introduced in the hybrid gene neighboring several pseudogene-specific point mutations.

As expected from a phenotype including adrenal insufficiency, newborn aw18 homozygous mice (H-2aw18) did not survive the early postnatal stage. Furthermore, affected animals presented with evident morphological changes in the adrenal glands, indicating adrenocortical hyperplasia [33]. Therefore, in addition to various ste-roid substitution regimens to prolong survival, H-2aw18 animals were also utilized to study therapeutic strate-gies such as transgenic rescues through overexpression of Cyp21 [34]. Using this approach, prolonged survival without further substitution could be achieved in vari-ous proportions of the transgenic animals (between 20% and 80% depending on the integration of the transgene). Finally, H-2aw18 animals were utilized as a model of adre-nal insufficiency to dissect the interrelationship between adrenal cortical and medullary function. Specifically, it could be demonstrated that catecholamine secretion is severely affected in Cyp21-deficient animals [35]. Of note, similar functional alterations between the model organism and affected patients were appreciated only after implementation of dedicated clinical protocols [36].

13A. ANIMAL MODELS OF ADRENAL GENETIC DISORDERS326

11β-Hydroxylase Deficiency

Patients with CAH due to mutations of the 11β-hydroxylase gene (CYP11B1), the final enzyme in the glucocorticoid biosynthetic pathway, are characterized by glucocorticoid deficiency, adrenal hyperplasia driven by unsuppressed hypothalamo–pituitary– adrenal activity, which are shunted into the adrenal androgen synthesis pathway, and excess min-eralocorticoid activity caused by the accumulation of deoxycorticosterone [37].

Specific CAH mouse model animals were created by targeted replacement of Cyp11b1 with a gene for a fluo-rescent protein. As expected, the urinary steroid profile of Cyp11b1 knockout mice was similar to that of patients carrying null mutations of the 11β-hydroxylase gene with evidence of glucocorticoid depletion and miner-alocorticoid and progesterone excess [38]. Through a 30-fold increase in deoxycorticosterone its weak miner-alocorticoid activity resulted in a significant hypokale-mia, decrease in plasma renin concentration, and lower levels of aldosterone in affected mice. Furthermore, adrenal hyperplasia was evident in homozygous knock-out animals. Thereby, this mouse model resembles many aspects of the clinical phenotype seen in patients with CAH and could aid in future mechanistic or therapeutic studies.

MOUSE MODELS OF CONGENITAL LIPOID ADRENAL HYPERPLASIA

StAR Deficiency

Not long after cloning and description of steroido-genic acute regulatory protein (StAR) as the initial step in adrenal and gonadal steroidogenesis [39], a mouse model was generated upon targeted disruption of the mouse Star gene [40]. Based on the clinical presentation of affected patients with various degrees of adrenal insuf-ficiency, it had been hypothesized that two hits contrib-ute to congenital lipoid adrenal hyperplasia [41]. First, a mutant StAR prevents the acute steroidogenic response in the adrenal gland and gonad during development. At this point, some baseline steroidogenesis independent of StAR activity might still occur. Second, the accumulation of cholesterol esters and sterol auto-oxidation products in affected cells results in cellular damage and eventu-ally disrupts the basal steroidogenesis that is indepen-dent of steroidogenic acute regulatory protein.

Interestingly, similar to the clinical situation, pheno-typic variability was also observed in the Star knock-out mouse model as survival of untreated homozygous animals ranged between 1 and 16 days after birth [42]. Morphologically, adrenal cortices of Star knockout mice contained lipid deposits very similar to what had been

described in patients with congenital lipoid adrenal hyperplasia. To extend the phenotypic characterization of knockout animals, steroid replacement therapy was implemented to increase the lifespan of affected mice. In this treatment group progressive increases in lipid deposits were evident [43]. In addition, the gonadal phe-notype, which was only mild in newborn animals, was accelerated over time, with progressive histopathologi-cal changes that occurred with aging.

Star knockout animals were also utilized for trans-genic rescue experiments with green fluorescent protein (GFP)-tagged wild-type StAR sequences in comparison to those lacking a mitochondrial targeting signal [44]. Earlier in vitro experiments in cell systems had suggested that expression of the truncated StAR protein stimulated steroidogenesis at protein levels comparable to those seen normally in steroidogenic cells [45]. In contrast, in the in vivo setting the mutated StAR protein expressed in the context of otherwise Star-deficient mice was able to rescue the lethal phenotype in only 40% of cases [44]. The same transgenic in vivo system was utilized to assess changes in molecular patterns that are associ-ated with morphological alterations in the adrenals of Star knockout mice. As demonstrated by transcriptome analysis from sorted adrenal cells, expression levels of genes involved in cholesterol efflux and the inflamma-tory response were upregulated in knockout animals whereas those related to steroidogenesis or cholesterol biosynthesis and influx were not significantly altered [46]. Therefore, it is likely that excessive accumulation of intracellular cholesterol induced by Star deficiency results in cellular responses to counteract this alteration to maintain the integrity of the steroid-producing cell. Therefore, it has been hypothesized that these compen-satory mechanisms might be one of the reasons for the initially preserved functional capacity of steroidogenic cells in animals and patients with Star deficiency which is abrogated at a later time point by further structural changes.

Side Chain Cleavage Enzyme Deficiency

In addition to StAR, mutations in the side chain cleav-age enzyme encoded by CYP11a1 have been attributed to be responsible for the development of congenital lipoid adrenal hyperplasia. As CYP11a1 is important for placental progesterone synthesis and progesterone is essential for the maintenance of human pregnancy, complete loss of Cyp11a1 activity had been predicted to be incompatible with life. However, severe cases with homozygous disruption of Cyp11a1 activity have been described, with early adrenal insufficiency and com-plete 46,XY sex reversal [47]. In less severe forms adre-nal insufficiency occurred only later in life, with normal male genital development [48]. Heterozygous carriers

IonS InDuCInG ADREnAl TuMoRS 327

MouSE MoDElS wITH TARGETED DElET

of Cyp11a1 mutations have been reported to be without apparent clinical symptoms.

Similar to the clinical situation, heterozygous Cyp11a1-deficient mice (upon generation by gene targeting with the neo gene inserted into the first exon of Cyp11a1) were demonstrated to be fertile and apparently normal [49]. Furthermore, homozygous knockout mice were born without embryonic lethality, indicating that Cyp11a1 is not essential for survival of mouse embryos. However, most likely because of adrenal insufficiency, most homo-zygous knockout null mice were reported to die within 1–2 days after birth and few survived up to 7 days. In another mouse model only the Sf1 response element of the Cyp11a1 promotor was mutated, which resulted in a less severe phenotype with reduced stress response owing to decreased adrenal Cyp11a1 expression and insufficient stress-induced glucocorticoid secretion [50]. Furthermore, these animals, which express only 15% of the normal amounts of Cyp11a1 in their adrenals, had a normal lifespan and were found to develop adrenal hyperplasia with progressive increase in adrenal weight during aging.

Very similar to the phenotype in Star knockout ani-mals, Cyp11a1-deficient mice start to aggregate lipid depots in their adrenals during development which eventually leads to cell damage. However, electron microscopic analyses of newborn mice revealed that the mitochondria in Star knockout mice were gener-ally less perturbed than those of Cyp11a1 knockout animals [51]. Based on these ultrastructural findings it was hypothesized that Star deficiency, with its florid lipid deposits within the cell and relative sparing of the mitochondria itself, is associated with some pres-ervation of steroidogenic capacity owing to StAR-independent cholesterol transfer. In contrast, in the context of Cyp11a1 deficiency, where adrenal cells com-pletely lack the enzyme that catalyzes the initial step of steroidogenesis, this would result in a more severe structural mitochondrial phenotype. Thus, these data provided the first insights into potential differences in the pathogenesis of these two forms of lipoid congeni-tal adrenal hyperplasia.

GENETICALLY MODIFIED MOUSE MODELS DISPLAYING AN ADRENAL

TUMOR PHENOTYPE

Mice that have been designed to harbor specific genetic modifications through transgenic techniques or knockout approaches have been instructive for the iden-tification of molecular mechanisms involved in adreno-cortical tumorigenesis. These models can be utilized to provide information on the functional significance of a specific gene or downstream pathway that might have

been identified by in vitro experiments, through expres-sion studies from surgical tumor samples, or on the basis of clinical information from patients with rare genetic syndromes. Furthermore, careful phenotypic character-ization of available mouse models in which an adrenal phenotype has been discovered can serve as a starting point for further functional analysis.

MOUSE MODELS WITH TARGETED DELETIONS INDUCING ADRENAL

TUMORS

An example in which the occurrence of adrenocorti-cal carcinoma (ACC) is indicative of the presence of a genetic syndrome is that of childhood adrenal cancer in the context of specific TP53 mutations [52]. p53 acts as a cell cycle checkpoint to regulate DNA repair or induce growth arrest or apoptosis in response to DNA damage, and its loss of function has been demonstrated to affect a large array of tumor entities [53]. However, only very recently has its role as a tumor suppressor gene in ACC been highlighted in a mouse model of telomere dys-function in which animals with p53 haploinsufficiency developed ACC in 5% of cases [54]. While these tumors exhibited locally invasive growth and a malignant his-tology, no metastatic spread has been reported.

In patients with multiple endocrine neoplasia type 1 (MEN1), in addition to parathyroid adenomas, pan-creatic islet tumors, and pituitary adenomas, develop-ment of adrenocortical tumors has been described in up to 40% of patients [55]. Accordingly, animals with targeted deletion of the menin gene resembled the clini-cal features of MEN1, including that of development of adrenocortical nodular disease which progressed into adrenal tumors [56–58]. As part of an aging experiment, adrenocortical lesions described as microadenomas or tumors developed in 6% of heterozygous animals within the first year of life, and in up to 30% in a cohort of roughly 2-year-old animals [58]. In addition to these small lesions, adrenal tumors with a more aggressive growth behavior have been reported, with an incidence after 18 months of up to 46% in heterozygous animals [57]. Notably, other MEN1-defining tumors, including pancreatic islet cell tumors and pituitary adenomas, developed at an earlier time point and with higher penetrance [56,58]. As homozygous menin knockout animals die in utero, only heterozygous mice were phenotypically characterized. However, in accordance with a two-hit model of a tumor suppressor gene, the remaining wild-type menin allele could be demon-strated to be lost in somatic tumor cells [56–58]. This is in striking contrast to human adrenal lesions as part of MEN1, where loss of heterozygosity does not seem to be a dominant tumorigenic mechanism.

13A. ANIMAL MODELS OF ADR328

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