Adrenocortical Development, Maintenance, and Disease
Rebecca Yates*, Harshini Katugampolat, Dominic Cavlan+, Katy Cogger1, Eirini Meimaridout, Claire Hughes+, Louise Metherell+, Leonardo Guasti+, Peter King+, +,1
*Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, London, United Kingdom
*Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, London, United Kingdom
1Corresponding author: e-mail address: p.j.king@qmul.ac.uk
Contents
| 1. Introduction | 240 |
| 2. The Adrenal Gland: Structure and Function | 241 |
| 2.1 Anatomy | 241 |
| 2.2 Blood supply | 243 |
| 2.3 Nerve supply | 243 |
| 2.4 Steroid synthesis | 244 |
| 2.5 Glucocorticoid synthesis and function | 245 |
| 2.6 Aldosterone synthesis and function | 247 |
| 2.7 Catecholamine synthesis and function | 248 |
| 3. Adrenal Development | 249 |
| 3.1 Early adrenal development | 249 |
| 3.2 Early medulla development | 250 |
| 3.3 Later development | 251 |
| 3.4 Neonatal period and adult growth | 252 |
| 4. Theories of Growth and Zonation | 252 |
| 5. Factors Involved in Adrenal Development | 254 |
| 5.1 Urogenital ridge development | 254 |
| 5.2 AGP development | 256 |
| 5.3 Adrenal primordium development | 259 |
| 5.4 Transcriptional control of SF-1 | 261 |
| 6. Signaling Pathways Involved in Adrenal Development | 262 |
| 6.1 Shh signaling | 262 |
| 6.2 FGF signaling | 264 |
| 6.3 Fetal adrenal growth | 265 |
| 6.4 Fetal zone involution and X zone regression | 266 |
| 7. Models of Development | 266 |
| 7.1 SF-1 FAdE | 266 |
| 7.2 Shh/Gli1 lineages | 268 |
| 7.3 Stem/progenitor cells | 270 |
| 8. Control of Postnatal Growth, Zonation, and Remodeling | 272 |
| 8.1 Normal growth | 272 |
| 8.2 Zonation | 273 |
| 8.3 Remodeling | 274 |
| 8.4 Regeneration following surgical injury | 274 |
| 8.5 ACTH and remodeling | 275 |
| 9. Diseases of the Adrenal Cortex | 277 |
| 9.1 IMAGe | 278 |
| 9.2 Familial glucocorticoid deficiency | 278 |
| 9.3 Triple-A syndrome | 281 |
| 9.4 Adrenal cancer | 282 |
| 10. Summary | 291 |
| References | 292 |
Abstract
The adrenal gland controls a plethora of crucial physiological functions, and dysfunction is associated with severe morbidity. Because of the vital importance of appropriate adre- nal function, the development and function of the gland have been intensively studied, and these investigations have revealed fascinating developmental origins and a remark- able remodeling and regenerative capacity in the adult. This chapter, focusing on the adrenal cortex, will describe our current understanding of the development and main- tenance of the adrenal gland, which has been advanced over recent years by the use of sophisticated genetic models in the study of both normal function and disease. This work has shed light on the transcriptional networks and signaling pathways involved in development and maintenance of the gland and in its pathology; these are discussed in the light of the wealth of physiological information gathered in studies of human and rodent adrenal development and function.
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1. INTRODUCTION
The earliest descriptions of the adrenal gland were made by Galen in the second century, but the gland was definitively described and illustrated by in 1563 by Eustachio (1563) who recognized that the structures he observed were discrete organs, albeit of unknown function. It was not until nineteenth-century advances in lens manufacture and microscopy that Kölliker (1852) could distinguish the cortex from the medulla and conclude on the basis of their separate early development and different appearance and that these two structures had different functions. Thomas Addison was the first to recognize the importance of the adrenal gland for well-being
(Addison, 1855) and Charles-Edouard Brown-Sequard demonstrated in 1856 that experimental animals could not survive bilateral adrenalectomy (Brown-Sequard, 1856). The cortical zones were later identified and named by Arnold (1886). The adrenal gland has been the subject of continuous investigation since these discoveries, and the chapter will discuss the phys- iology of the gland, in particular the cortex, and more recent mechanistic insights into development, growth, maintenance, and disease of this organ.
2. THE ADRENAL GLAND: STRUCTURE AND FUNCTION
2.1. Anatomy
The adrenal glands are a pair of composite endocrine organs, with the outer cortex and inner medulla having different functions and developmental ori- gins. The glands are located anterior to and superior to the upper pole of the kidney (Fig. 7.1). They are yellowish in color and asymmetrical, with the right adrenal gland being pyramidal in shape and the larger left adrenal gland being crescent-shaped. Adult human adrenal glands weigh 8-10 g, up to 90% of which is cortex. The adrenal cortex is the principal steroid- producing organ in the body and the medulla secretes catecholamines that function as “fight or flight” hormones.
The adrenal gland is enclosed within a capsule of fibroblasts and myo- fibroblasts, which contains an arterial plexus and sends trabeculae containing blood vessels and nerves into the underlying cortex. Capsular cells are flat- tened with elongated nuclei arranged in the plane of the capsule. Lying underneath the capsule is the outermost zone of the cortex, the zona glomerulosa (ZG). This narrow zone contains relatively small columnar epi- thelial cells arranged in round clusters. The ZG cells have intensely staining nuclei and basophilic cytoplasms and have low lipid content. Their mito- chondria are typified by the lamelliform ultrastructural appearance of their cristae. This zone constitutes up to 15% of the cortex and produces the min- eralocorticoid aldosterone, responsible for controlling blood volume and salt/water balance. Beneath the ZG is the zona fasciculata (ZF), which con- tains lipid droplet-rich polygonal epithelial cells arranged in radial columns separated by fenestrated capillaries. The mitochondria of these cells have tubulovesicular cristae. The ZF predominantly secretes glucocorticoids- cortisol in humans and corticosterone in mice and rats-which have influ- ential effects on metabolism, the cardiovascular system, and the immune system. The innermost cortical zone is the zona reticularis (ZR), made up of polyhedral cells with prominent lysosomes arranged in a mesh-like
Cortex
Medulla
Capsule ZG
Capsule ZG
Capsule
ZG
oZU
ZF
İZU
ZF
ZF
ZR
X zone
Medulla
Medulla
Medulla
Human adrenal
Mouse adrenal
Rat adrenal
structure of anastomosing cords. The ZR makes glucocorticoids and, in humans and higher primates, adrenal androgens such as androstenedione, dehydroepiandrosterone (DHEA), and its sulfate, DHEAS. The cortical zones were identified and named by Arnold (1886).
The medulla makes up the inner mass of the gland and is composed of large, polygonal, epithelial cells surrounded by venous sinusoids, and these cells produce catecholamines, not only principally adrenaline but also some
noradrenaline depending upon the species. There is minimal, if any, inter- vening connective tissue between the cortex and the medulla, leaving cor- tical and medullary cells in direct contact.
Although the general arrangement of the gland is the same across mam- malian species, some differences in zonation can be observed, particularly between those most studied, humans, mice, and rats. In the rat, the zones are organized in concentric shells, with the ZG a continuous zone beneath the capsule. However, the ZG does not immediately abut the ZF. Instead, there is a sudanophobic zone three to four cells thick referred to variously as the zona intermedia, white zone, or undifferentiated zone (ZU) (Cater & Lever, 1954; Guasti, Paul, Laufer, & King, 2011; Mitani et al., 1994). These cells have mitochondria similar to those of the ZF with tubulovesicular cris- tae (Domoto, Boyd, Mulrow, & Kashgarian, 1973). In the mouse, however, there is no UZ and the innermost ZG and outermost ZF cells directly con- tact each other (King, Paul, & Laufer, 2009; Paul & Laufer, 2011). The human ZG is arranged in discrete clusters such that some cords of ZF extend between them as far as the capsule (Boulkroun et al., 2010, 2011; Nishimoto et al., 2010) (Fig. 7.1).
2.2. Blood supply
Superior, middle, and inferior adrenal arteries arise from the aorta, renal, and inferior phrenic arteries and ramify over the capsule before forming a sub- capsular arteriolar plexus. From here, capsular capillaries feed thin-walled venous sinusoids that traverse the cortex and form another plexus in the ZR in the adult, while venules pass between the medulla chromaffin cells to enter the adrenal vein. The fully formed medulla is also supplied by direct branches from the subcapsular plexus, which bypass the ZR plexus. This serves to ensure that most cells in the adrenal gland are only one to two cells away from a vascular endothelial cell, resulting in efficient delivery of adrenal hormones to the blood stream. Venous drainage from the adrenal gland is usually via a single vein, directly to the inferior vena cava on the right and the renal vein on the left. There are subcapsular and medullary lym- phatic plexus, draining to lumbar and para-aortic lymph nodes (Dobbie & Symington, 1966).
2.3. Nerve supply
The autonomic nerve supply of the adrenal gland is the largest of any organ, relative to its size. Myelinated sympathetic preganglionic fibers from the
intermediolateral cell column or lateral horn of T10-11 synapse on the med- ullary chromaffin cells. The cortex receives afferent nerve supply from the medulla, with sympathetic fibers traveling along the blood vessels (Parker, Kesse, Mohamed, & Afework, 1993).
2.4. Steroid synthesis
The adrenal steroids are synthesized from cholesterol by a series of reactions that are catalyzed by two classes of enzymes, cytochrome P450 (CYP) mixed function oxidases and short-chain dehydrogenases, and take place in the inner membrane of mitochondria or in the smooth endoplasmic reticulum (SER) with the intermediate compounds shuttling back and forth (for review, see Payne & Hales, 2004). Cholesterol, principally obtained in ester- ified form from LDL (humans) and HDL (rodents) via LDL receptor endocytic or SR-B1 selective transport pathways, respectively (for review, see Hu, Zhang, Shen, & Azhar, 2010), is initially cleaved from its esterified form by cholesteryl ester hydrolase and translocated from the outer to the inner mitochondrial membrane by the transporter enzyme steroidogenic acute regulatory protein (StAR). This occurs preferentially at sites of close contact between the membranes that are more numerous in tubulovesicular than lamelliform mitochondria (Vinson, 2003). The preponderance of tubulovesicular mitochondria in the ZF compared to the ZG reflects the higher levels of steroidogenesis in the ZF than the ZG. Once translocated, cholesterol is converted by side-chain cleavage (CYP11A1) into pregneno- lone. This is then transported to the SER where, in humans, it is converted to 17OH pregnenolone by CYP17 (17a-hydroxylase) in the ZF and ZR but not in the ZG, where CYP17 is not expressed. 30-hydroxysteroid dehydrogenase (3ßHSD) converts 17OH pregnenolone to 17OH proges- terone in the ZF and ZR and pregnenolone to progesterone in the ZG. Still in the SER, CYP21 (21-hydroxylase) converts progesterone and 17OH progesterone to 11-deoxycorticosterone and 11-deoxycortisol, respectively. These compounds are transported back to the inner mitochondrial mem- brane, and CYP11B1 (11ß-hydroxylase) converts 11-deoxycortisol to cortisol, the major human glucocorticoid, in the ZF. In the ZG, 11-deoxycorticosterone is converted to aldosterone, the major mineralocor- ticoid, by CYP11B2 (aldosterone synthase). CYP17 is not expressed in the adult cortex in rats and mice, and 11-deoxycorticosterone is the substrate for both CYP11B2 and B1, resulting in corticosterone rather than cortisol being the glucocorticoid produced in these species. CYP17 has two activities,
acting as both a 17x-hydroxylase and a 17,20-lyase. The hydroxylase activity is required for cortisol production but cytochrome b5, expressed in the ZR, promotes the lyase activity that cleaves two carbon atoms to produce DHEA from 17OH pregnenolone, such that cortisol production is suppressed in the ZR and adrenal androgens are formed instead (Endoh, Kristiansen, Casson, Buster, & Hornsby, 1996). DHEA can then be sulfated by SULT2A1 to DHEAS or converted to androstenedione by 3ßHSD. These are the major androgens produced in the human adrenal gland but small amounts of tes- tosterone can also be produced from androstenedione by 17-ketosteroid reductase (17ßHSD) (Fig. 7.2). Because adult rat and mouse adrenal glands do not express CYP17, they do not synthesize adrenal androgens and the ZF and ZR are considered to be functionally equivalent.
2.5. Glucocorticoid synthesis and function
Glucocorticoid production is under the control of the hypothalamic- pituitary-adrenal (HPA) axis. Upon activation of the HPA axis, parvocellular neurons of the hypothalamic paraventricular nucleus synthe- size and secrete corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) into the hypophyseal portal circulation (Carrasco & Van de Kar, 2003; Chrousos, 1995; Sapolsky, Romero, & Munck, 2000; Van de Kar & Blair, 1999). CRH is the main regulator of the release of adre- nocorticotropic hormone (ACTH) from the anterior lobe of the pituitary gland into the systemic circulation (Carrasco & Van de Kar, 2003). AVP has a synergistic role in CRH-induced ACTH release but is ineffective in the absence of CRH (Chrousos, 1995). CRH neurons projecting from the paraventricular nucleus of the hypothalamus onto proopiomelanocortin (POMC)-containing neurons in the hypothalamic arcuate nucleus can also stimulate ACTH production. They stimulate POMC release, which is cleaved to form ACTH (Pritchard & White, 2007).
ACTH binds to the ACTH receptor MC2R (melanocortin receptor 2), a 7-transmembrane G protein-coupled receptor (GPCR), causing the a-subunit of the stimulatory heterotrimeric G protein (Gas) to associate with adenylate cyclase. This catalyzes the conversion of ATP to cAMP, leading to the activation of downstream signaling pathways, including the activation of the cAMP-dependent protein kinase (PKA). PKA phosphorylates and acti- vates cholesteryl ester hydrolases and StAR, increasing the amount of cho- lesterol delivered to the inner mitochondrial membrane. cAMP also induces the transcription of StAR and CYP11A1, and CYP17 to promote cortisol
-
O
OH
“H
HO
H
H
H
H
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HO
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Cholesterol
CYP11B1
Corticosterone
CYP11A1
OH
OH
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o
O
O
O
H
3฿HSD
H
CYP21
H
CYP11B2
HO
H
H
H
H
H
H
H
H
H
HO
O
o
O
Pregnenolone
Progesterone
11-Deoxycorticosterone
Aldosterone
CYP17
CYP17
o
OH
O
O
OH
O
“OH
CYP21
OH
OH
3₿HSD
CYP11B1
HO
OH
H
H
H
H
H
H
H
H
H
H
H
H
HO
O
O
17OH-Pregnenolone
17OH-Progesterone
11-Deoxycortisol
Cortisol
CYP17
Cytb5
CYP17
Cytb5
O
o
OH
“OH
H
3ßHSD
H
17ßHSD
H
H
H
H
H
H
H
O
O
O
Dehydroepiandrosterone
Androstenedione
Testosterone
production (Aumo, Rusten, Mellgren, Bakke, & Lewis, 2010; Sewer, Dammer, & Jagarlapudi, 2007; Waterman & Bischof, 1996). Glucocorti- coids inhibit the secretion of CRH and AVP by the hypothalamus and POMC cleavage in the pituitary gland, thus reducing the production of ACTH and therefore cortisol itself in a negative feedback loop (de Kloet, 1995).
Cortisol is the principal glucocorticoid produced by the human ZF, with influential effects on metabolism, the cardiovascular system, and the immune system. Although at basal levels its role is a predominantly permissive one, allowing the effects of other agents, it is released in response to stress. Glu- cocorticoids increase blood glucose levels by stimulating gluconeogenesis, inhibiting glucose storage, and stimulating lipolysis in adipose tissue to release free fatty acids. They can also cause proteolysis in some muscle tissues. In the cardiovascular system, cortisol increases the transcription of receptors for angiotensin II (AngII), epinephrine, and norepinephrine, to regulate blood pressure, contractility, and tone of the heart (Sakaue & Hoffman, 1991).
Glucocorticoids also have important anti-inflammatory properties, inhibiting the actions of immune cells (T and B lymphocytes) and the synthesis and release of cytokines (including interleukins (IL-)1-6, IL-12, IFN-Y, TNF-a, and chemokines) and other inflammatory mediators, such as histamine and bradykinin. In this way, cortisol prevents excessive inflam- mation and tissue and organ damage (Franchimont, 2004).
2.6. Aldosterone synthesis and function
Aldosterone production is induced by increases in extracellular potassium levels and by ACTH, although AngII is the primary regulator, via the renin-angiotensin-aldosterone system (RAAS). The RAAS has little con- trol over arterial blood pressure under normal circumstances but plays a piv- otal role when sodium levels fall or hemorrhage occurs, as it has major vasoconstrictor capabilities (Collier, Robinson, & Vane, 1973; Scornik & Paladini, 1964). Renin is a proteolytic enzyme that cleaves angiotensinogen, synthesized by the liver, into angiotensin I (Miller, 1981). It is released from the juxtaglomerular cells of the kidney into the blood, stimulated by sym- pathetic nerve inputs, catecholamines, decreased renal perfusion pressure, and decreased sodium delivery to the distal tubule (Johnson & Davis, 1973; Tobian, Tomboulian, & Janecek, 1959). Angiotensin I is hydrolyzed by angiotensin-converting enzyme (ACE), found at the surface of
pulmonary and renal endothelium, forming AngII (Oparil, Sanders, & Haber, 1970). This provokes vasoconstriction of arteriolar smooth muscle, increases the contraction of the heart (positive inotropic effect), elicits the release of catecholamines, and stimulates aldosterone production by the ZG of the adrenal cortex.
AngII binds the AT1 cell surface receptor expressed on ZG cells, which is a GPCR coupled to phosphoinositidase C (PI3C) (Bird et al., 1993). PI3C causes hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating ino- sitol 1,4,5-trisphosphate (Ins(1,4,5)P3) and 1,2-diacylglycerol (DAG). Ins(1,4,5)P3 is an intracellular second messenger that opens calcium channels on intracellular stores, resulting in the release of calcium into the cytoplasm (Wojcikiewicz & Nahorski, 1993). Elevated intracellular calcium levels acti- vate calmodulin and CaMK (calmodulin-dependent protein kinases), and DAG activates protein kinase C (PKC), leading to the phosphorylation and activation of other second messenger cascades, which modulate aldoste- rone production, possibly by phosphorylation of transcription factors regu- lating CYP11B2 transcription (Nogueira, Bollag, & Rainey, 2009; Pezzi, Clyne, Ando, Mathis, & Rainey, 1997).
Aldosterone is the primary mineralocorticoid produced by the ZG of the adrenal cortex, regulating sodium retention, water balance, and blood pres- sure. Sodium is actively reabsorbed in the distal nephron of the kidney via ENaC sodium channels, whose activity is upregulated by aldosterone. This is accompanied by passive water reabsorption to increase the extracellular and blood fluid volumes and hence blood pressure (Miller, 1981). Aldoste- rone also has effects on the cardiovascular system and can cause cardiac fibro- sis due to the activation of an inflammatory cascade (Fuller & Young, 2005; Wehling et al., 1998).
2.7. Catecholamine synthesis and function
Tyrosine is converted to L-DOPA and then dopamine in medullary chro- maffin cells by the successive actions of tyrosine hydroxylase (a mixed func- tion oxidase, which can also hydroxylate phenylalanine to produce tyrosine) and DOPA decarboxylase (Fukami, Haavik, & Flatmark, 1990). Dopamine B-hydroxylase, another mixed function oxidase, then converts dopamine to noradrenaline, and this is converted to adrenaline by phenylethanolamine N-methyltransferase (PNMT) in adrenergic chromaffin cells. Dopamine, noradrenaline, and adrenaline are stored in vesicles along with chromogranin A. In humans, most chromaffin cells express PNMT, and therefore, almost
all the catecholamine output is adrenaline, but in rodents, some chromaffin cells do not express PNMT and approximately 20% is in the form of nor- adrenaline (Schinner & Bornstein, 2005; Wong, 2003).
The catecholamines bind to adrenergic receptors, with noradrenaline binding preferentially to a-receptors, while adrenaline binds to both a- and ß-receptors. Activation of these receptors causes rapid and diverse effects including vasoconstriction, piloerection increased heart rate, and increased blood glucose, all part of the “fight or flight” response.
X 3. ADRENAL DEVELOPMENT
3.1. Early adrenal development
The adrenal cortex develops from a thickening of the coelomic epithelium located at the base of the groove between the urogenital ridge and the dorsal mesentery that can be identified in humans from about 4 weeks post- conception (wpc) (Else & Hammer, 2005; Parker et al., 2002) and 9.5-10 days postconception (dpc) in the mouse (Hatano, Takakusu, Nomura, & Morohashi, 1996; Ikeda, Shen, Ingraham, & Parker, 1994), referred to as the adrenogonadal primordium (AGP), a group of cells identified by their expression of the transcription factor steroidogenic factor-1 (SF-1, Ad4BP, and NR5A1) (Hatano et al., 1996; Luo, Ikeda, & Parker, 1994) (for reviews, see Else & Hammer, 2005; Kim et al., 2009; Laufer, Kesper, Vortkamp, & King, 2012) (Fig. 7.3). As this primordium grows, the cells delaminate from the coelomic epithelium and invade the underlying mesenchyme, with the cells adjacent to the mesonephros migrating dorsolaterally to form the gonadal primordium, while the more medial cells that express the highest levels of SF-1 migrate and condense to form the adrenal primordium at the cranial end of the mesonephros, ventrolateral to the dorsal aorta. This occurs at 33 dpc (Goto et al., 2006; Hanley, Rainey, Wilson, Ball, & Parker, 2001) (10.5 dpc in the mouse). The cells of the adrenal primordium continue to proliferate and CYP17 expression is detectable from 41 to 44 dpc (Goto et al., 2006; Hanley et al., 2001) with vascularization of the gland apparent at this time. By 50-52 dpc, two discrete zones are detectable in the adrenal cortex, the inner zone, referred to as the fetal zone (FZ), com- posed of large polyhedral eosinophilic cells with tubulovesicular mitochon- drial cristae with high levels of expression of steroidogenic enzymes, and the smaller outer zone, or definitive zone (DZ), which is composed of small, tightly packed basophilic cells (Goto et al., 2006) with lamelliform mito- chondrial cristae with much lower levels of steroidogenic enzyme
A
B
SA
C
Capsule
V
NT
SA
AP
Ch
Nº DA
%
CE
GUT
AGP
GP
Developing adrenal
expression (Goto et al., 2006; Hanley et al., 2001; Nussdorfer, 1986). The periphery of the gland is now heavily vascularized and the mesenchymal capsule is forming at this time in human adrenal development and is com- plete by the 9th week.
3.2. Early medulla development
The adrenal medulla develops from the trunk neural crest. Some of these cells migrate ventrally through the anterior sclerotome to reach the dorsal aorta and form the sympathoadrenal (SA) lineage (Anderson, Carnahan, Michelsohn, & Patterson, 1991). This sympathetic primordium gives rise to tyrosine hydroxylase-expressing catecholaminergic neuronal progenitor cells in response to bone morphogenetic protein cues from cells of the wall of the dorsal aorta and the surrounding mesenchyme (Reissmann et al., 1996; Shah, Groves, & Anderson, 1996). Some cells then migrate from the dorsal aorta along nerves and blood vessels to enter the adrenal primor- dium at the cranial end from 6 wpc (Ehrhart-Bornstein et al., 1997; Yamamoto, Yanai, & Arishima, 2004) and acquire the phenotype of chro- maffin cells. It was thought that these were SA cells that lost expression of neuronal genes, but more recently, it has been shown that at least some of the presumptive chromaffin cell population enters the adrenal primordium still expressing neural crest markers and then gains TH expression but not
neuronal markers (Ernsberger et al., 2005). Initially, the chromaffin cells are scattered within the cortex and are mostly noradrenergic, until 10 wpc when small islets of chromaffin cells express PNMT, the enzyme required to con- vert noradrenaline into adrenaline, to become adrenergic (Jozan et al., 2007).
3.3. Later development
By 9 wpc, the human adrenal gland is encapsulated with a cortex containing an outer DZ and inner FZ and islands of chromaffin cells in the center and has become heavily vascularized with adrenal arteries supplying blood to a subcapsular arteriolar plexus, and these give rise to capsular capillaries that feed thin-walled venous sinusoids and traverse the cortex. The gland con- tinues to grow rapidly, increasing approximately 8- to 10-fold in size from this stage until birth, largely due to an increase in the size of the FZ, which accounts for 80-90% of the weight of the gland by midgestation (Keene, 1927; Spencer, Mesiano, Lee, & Jaffe, 1999), and a third cortical zone becomes evident by 14 weeks (Goto et al., 2006; Sucheston & Cannon, 1968). This transitional zone (TZ) (Mesiano, Coulter, & Jaffe, 1993; Mesiano, Mellon, & Jaffe, 1993) is located between the DZ and the FZ and contains cells whose histological appearance shares features of both DZ and FZ. By late gestation, the DZ has begun to resemble the ZG and the TZ, the ZF (Coulter & Jaffe, 1998; Sucheston & Cannon, 1968).
Steroid production from the fetal adrenal gland has important roles in maintaining intrauterine homeostasis and in the maturation of fetal organ systems (including the adrenal gland itself) in preparation for extrauterine life. The human placenta cannot make estrogens de novo, but the large amounts of DHEA and DHEAS produced by the FZ (100-200 mg per day; Carr & Simpson, 1981) are converted by placental aromatase to estro- gens, which maintain pregnancy. The TZ produces cortisol, with an early peak from 8 to 9 wpc coinciding with transient 3ßHSD expression (Goto et al., 2006). This is thought to protect female sexual development by neg- atively regulating the fetal HPA axis and thus inhibiting adrenal androgen production during this developmental window. Immediately before birth, there is a second peak in cortisol production that is required for fetal organ maturation, and this is at least partly regulated by ACTH from the fetal pitu- itary gland. Cortisol from the TZ also induces PNMT expression in chro- maffin cells, thereby directing them to become adrenergic (Finotto et al., 1999; Wurtman & Axelrod, 1965).
3.4. Neonatal period and adult growth
Because of the rapid increase in the size of the FZ during gestation, the size of the neonatal human adrenal gland is relatively very large compared to the adult gland, weighing 3-5 g at term. After birth, the FZ rapidly involutes and remodels by a process involving apoptosis of cells in the inner region of the FZ (Jirasek, 1980), and there is a concomitant decrease in adrenal androgen secretion. The adult cortex develops from the DZ and TZ, giving rise to the ZG and ZF, respectively, and the weight of the adrenal gland drops by 50% within the first 2 weeks after birth. Cells with ZR morphol- ogy are detectable in humans from around 3 years of age until a continuous ZR forms at around 6 years of age and adrenal androgen synthesis recommences, a stage referred to as adrenarche. The ZR continues to increase in thickness until puberty (Hui et al., 2009; Nakamura, Gang, Suzuki, Sasano, & Rainey, 2009). After birth, the islands of chromaffin cells within the cortex coalesce to form a contiguous medulla (Wilburn, Goldsmith, Chang, & Jaffe, 1986).
In mice, there is a zone thought to be analogous to the FZ, namely, the X zone, next to the medulla. Unlike the FZ, however, the X zone starts to develop after birth with clusters of small lipid-poor cells with acidophilic cytoplasms appearing adjacent to the medulla. By weaning, the X zone has increased in size to occupy about one-third of the cortex, with a grada- tion of acidophilia from deep staining by the medulla to light staining adja- cent to the ZF. The X zone degenerates in males at puberty (~45 days), whereas in females, it undergoes vacuolar degeneration at first pregnancy or gradually degenerates in unbred females (Jones, 1948; Sucheston & Cannon, 1972).
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4. THEORIES OF GROWTH AND ZONATION
The adrenal cortex, as well as having a constant turnover of cells to replace dying cells, is a highly dynamic organ that undergoes rapid changes in response to hormonal demand and surgical injury, following which it shows remarkable regenerative capacity. Observations on adrenocortical cell growth from the late nineteenth century led to a number of theories of the origin of the cells of the different adrenocortical zones, but the weight of evidence favors the centripetal migration hypothesis, in which there is a continual production of new cortical cells at the periphery of the gland that migrate under this mitotic pressure to the inner cortical medulla boundary
where they die. This theory was initially advanced by Gottschau (1883), and evidence was subsequently provided by observing that the majority of mitotic cells in mice and rats were present at the boundary between the ZG and the ZF and that most cell death occurred at the ZR /medulla bound- ary (for a review, see Vinson, 2003). Early lineage-tracing studies performed by subcutaneous injection of trypan blue in rats revealed that, after initial uptake by the capsule and ZG cells, dye could be observed moving into the ZF and eventually be detected in cells in the ZR (Salmon & Zwemer, 1941). More recently, mosaic transgenic studies have provided supporting evidence for this theory, with columns of cells expressing B-galactosidase under the control of the Cyp21 promoter traversing the cor- tex from the capsule to the medulla boundary (Iannaccone, Morley, Skimina, Mullins, & Landini, 2003; Morley et al., 1996), and genetic lineage-tracing experiments have provided direct evidence for the centrip- etal migration (Huang, Miyagawa, Matsumaru, Parker, & Yao, 2010; King et al., 2009) (see later).
The precise origin of adrenocortical cells has been debated over the years. Zwemer, Wotton, and Norkus (1938) concluded from their intravital dye- labeling experiments that the newly synthesized cells were derived from the adrenal capsule, and this was supported by observations from enucleation studies in rodents (Baker & Baillif, 1939; Ingle & Higgins, 1939) (see later) and other remodeling protocols (Lombardo & Cortesini, 1988). However, others concluded from proliferation studies that the progenitor cells lie within the ZG (Mitani, Mukai, Miyamoto, Suematsu, & Ishimura, 2003; Taki & Nickerson, 1985). For example, the DZ cells possess characteristics of proliferating cells rather than steroidogenic cells; they express proliferating cell nuclear antigen (PCNA) and are relatively lipid-free and have mito- chondria with lamelliform cristae (McNutt & Jones, 1970; Sucheston & Cannon, 1968), and their arrangement as caps of the of FZ cells with some cells entering the outer FZ as columns has led to the suggestion that these are the progenitor cells of the ZF (Mesiano, Coulter, & Jaffe, 1993; Mesiano, Mellon, & Jaffe, 1993; Spencer et al., 1999). Many studies have identified proliferating cells in the rat adrenal cortex at the periphery of the gland between the ZG and the ZF (Bertholet, 1980; Ford & Young, 1963; Kataoka, Ikehara, & Hattori, 1996; Mitani, Mukai, Miyamoto, Suematsu, & Ishimura, 1999). Mitani and coworkers have demonstrated that the majority of proliferating cells are located around the UZ, which they have suggested is a stem cell zone in this species (Mitani et al., 1999, 2003). Nevertheless, it is generally agreed that cells arise at the periphery
and migrate through the cortex, with the implication that they change iden- tity as they move between zones.
5. FACTORS INVOLVED IN ADRENAL DEVELOPMENT
Insights into the mechanisms controlling adrenal development have come from the studies of abnormal adrenal development in both humans and animal models. Genetic analysis of human adrenal insufficiency and observations from knockout mouse models have identified genes that are involved in broadly three different areas of development: first, those that are involved in the development of the intermediate mesoderm and urogen- ital ridge and affect kidney, adrenal, and gonadal development; second, those that affect the development of the AGP and affect both gonadal and adrenal development; and third, those that more specifically affect the development of the adrenal gland.
5.1. Urogenital ridge development
5.1.1 Odd-skipped related 1
Odd-skipped related 1 (Odd1) is a transcription factor crucial for embryonic patterning and early development. It is expressed throughout the interme- diate mesoderm at 8.5 dpc and by 10.5 dpc in branchial arches and the limb buds (So & Danielian, 1999). Odd1-null mice die in utero, mostly between 11.5 and 12.5 dpc with no condensation of metanephric mesenchyme and impaired urogenital ridge formation, but those that survive to 15.5 dpc show complete absence of adrenal glands, gonads, and kidneys (Wang, Lan, Cho, Maltby, & Jiang, 2005).
5.1.2 WT1
The Wilms’ tumor gene WT1 is a Zn finger transcription factor involved in urogenital development and tumorigenesis that is expressed in the coelomic epithelium and the adjacent mesenchyme. Germline mutations in humans have been shown to cause defects in gonad and kidney formation (Baird, Groves, Haber, Housman, & Cowell, 1992; Haber & Housman, 1992). Wt1-null mice die at 13.5 dpc and gonad and kidney development does not extend beyond thickening of the coelomic epithelium (Kreidberg et al., 1993), but mice partially rescued by a human WT1 transgene survive to birth with urogenital abnormalities and much impaired adrenal develop- ment, indicating a role for WT1 in adrenal development (Moore, McInnes, Kreidberg, Hastie, & Schedl, 1999).
WT1 is expressed in the AGP and remains expressed in the bipotential gonad after separation from the adrenal primordium, but its expression is not observed in the adrenal primordium (Nachtigal et al., 1998; Val, Martinez-Barbera, & Swain, 2007).
5.1.3 Sall1
Sall1 is a member of the sal family of zinc finger-containing transcriptional regulators, and heterozygous mutations in this gene cause Townes-Brocks syndrome, which includes renal and genital abnormalities (Kohlhase, Wischermann, Reichenbach, Froster, & Engel, 1998). Sall1 is expressed in the intermediate mesoderm at 10.5 dpc and in the metanephric mesen- chyme surrounding the ureteric bud and mesonephros at 11.5 dpc (Nishinakamura et al., 2001) in mice and is expressed in the FZ of the human fetal adrenal gland (Ma et al., 2002). Sall 1-null mice have severe kidney dys- genesis or agenesis and hypoplastic adrenal glands at birth (Nishinakamura et al., 2001), whereas a mouse model that phenocopies the deletion seen in TBS has absent adrenal glands as well as kidneys and hypoplastic gonads at 16 dpc (Kiefer et al., 2003).
5.1.4 Pbx1
Pbx1 is a three-amino acid loop extension class homeodomain transcription factor that is essential for fetal development, with null mice dying at 15-16 dpc with multiple organ hypoplasia and aplasia (Selleri et al., 2001), including the kidney and adrenal gland (Schnabel, Selleri, & Cleary, 2003). At 10 dpc, Pbx1 is expressed in the nephrogenic cord and the coelomic epithelium, including the AGP (Zubair, Ishihara, Oka, Okumura, & Morohashi, 2006). By 14.5 dpc, Pbx1 is expressed throughout the urogenital structures, including the adrenal primordium, the interstitium of the gonads of both sexes, and the metanephric kidney (Schnabel et al., 2003). Pbx1 is detectable in human fetal adrenal glands by 5 weeks’ gestation and is expressed throughout the cortex by 10 weeks’ gestation (Ferraz-de- Souza et al., 2009). In Pbx1-null mice, by 12.0 dpc, the urogenital ridge is much reduced in size, and by 14.5 dpc, both the kidney and adrenal gland are absent, and gonadal development and sexual differentiation are poorly advanced (Schnabel et al., 2003). Pbx1+/- mice are viable and have smaller adrenal glands with a ZF containing fewer, but hypertrophied, cells and with fewer proliferative cells in the subcapsular region (Lichtenauer et al., 2007).
5.1.5 Wnt4
Wnt4 is a member of the Wnt family of secreted glycoproteins that have crucial roles in development and cell growth and differentiation. It is expressed in the metanephric mesenchyme on the medial side of the ureteric bud (Nishinakamura et al., 2001; Stark, Vainio, Vassileva, & McMahon, 1994) and the coelomic epithelium and mesenchyme of the genital ridge at 11.5 dpc in mice and is downregulated in the XY gonad from this time (Vainio, Heikkila, Kispert, Chin, & McMahon, 1999). By 14.5 dpc, its expression is localized in the subcapsular region of the adrenal primordium (Heikkila et al., 2002). A homozygous missense mutation in WNT4 in humans that results in greatly reduced WNT4 mRNA expression has been shown to be associated with renal agenesis, gonadal defects, and adrenal hypoplasia from 19 weeks’ gestation (Mandel et al., 2008), and Wnt4-null mice demonstrate impaired kidney development from 15 dpc (Stark et al., 1994) along with defects of the female reproductive system (Vainio et al., 1999), Sertoli cell differentiation in the testes (Jeays-Ward, Dandonneau, & Swain, 2004), and adrenal glands with reduced CYP11B2 expression and Pref-1/Dlk1 expression (Heikkila et al., 2002).
5.1.6 FoxD1/FoxD2
FoxD1 and FoxD2 are members of the forkhead/winged helix family of transcription factors that play important roles in controlling cell proliferation and differentiation during development. FoxD1 is expressed in the stromal mesenchyme surrounding the nephrogenic mesenchyme at 12.5 dpc in early kidney development (Hatini, Huh, Herzlinger, Soares, & Lai, 1996) and in the adrenal capsule at 14.5 dpc (Itaranta, Viiri, Kaartinen, & Vainio, 2009). FoxD2 is expressed in the intermediate mesoderm in mice and in the meta- nephric and mesonephric mesenchyme at 11.5 dpc, in the stromal cells at 12.5 dpc, and in the capsule of the developing adrenal gland at 15.5 dpc (Kume, Deng, & Hogan, 2000). Homozygous null FoxD1 and FoxD2 mice have hypoplastic adrenal glands at birth as well as developmental defects of the kidneys and ureter.
5.2. AGP development
5.2.1 SF-1
SF-1 is an orphan nuclear receptor transcription factor that is a key regulator of both steroidogenic organ development, namely, the gonads and the adre- nal glands, and steroidogenesis. It is expressed in all cells of the AGP, in both the DZ and FZ in humans, being the earliest detectable marker, with the
highest expression levels in the cells at the rostral end of the AGP, which will form the adrenal primordium (Hanley et al., 1999; Hatano et al., 1996; Ikeda et al., 1994). Following separation of the primordia, SF-1 expression is maintained in all steroidogenic cells of the adrenal cortex, but is not expressed in the medulla or the capsule. During sexual differentiation of the gonads, SF-1 expression remains high in the developing testes in both the testicular cords and the Leydig cell interstitial compartment but declines in midgestation in the developing ovaries until it is reestablished in steroido- genic cells in late gestation (Ikeda et al., 2001).
Absence of SF-1 function causes severe defects in both adrenal and gonadal development in both humans and mice (for review, see El-Khairi & Achermann, 2012). Homozygous deletion of the Sf-1 gene in mice causes loss of the adrenal glands and gonads by 12.5 dpc (Luo et al., 1994; Sadovsky et al., 1995). In Sf-1-null mice, the thickening of the coelomic epithelium that forms the AGP is observed but the cells undergo apoptosis by 11.5 dpc and the adrenal and gonadal primordia are lost by 12.5 dpc. Offspring die shortly after birth due to hypoglycemia as a result of adrenal insufficiency and exhibit XY sex reversal. Sf-1+/- mice are viable with normal gonadal development but have adrenal primordia with 12-fold smaller cross-sectional area at 12 dpc, which recovers to be only twofold smaller at 18.5 dpc, as a consequence of increased proliferation of the cortical cells from 13.5 dpc (Bland, Fowkes, & Ingraham, 2004). Adrenal glands in adult Sf-1+/- mice remain small (Babu et al., 2002) and have a reduced corticosterone output in response to stress (Bland et al., 2000). Heterozygous loss-of-function mutations have been identified in humans, but no cases of homozygous mutations have been reported, pre- sumably indicating the importance of SF-1 for human fetal development (El-Khairi, Martinez-Aguayo, Ferraz-de-Souza, Lin, & Achermann, 2011; Ferraz-de-Souza, Lin, & Achermann, 2011). In contrast to the situ- ation in mouse heterozygous knockouts, only two loss-of-function muta- tions have been reported that affect adrenal function, although this is a common cause of disorders of sex development in humans (Ferraz-de- Souza et al., 2011). The first was a patient who presented with adrenal failure and gonadal dysgenesis in infancy and was found to have a heterozygous G35E change in the DNA-binding domain of the protein (Achermann, Ito, Hindmarsh, & Jameson, 1999). Another mutation in the DNA-binding domain, R92Q, caused a similar phenotype but only when inherited homo- zygously, presumably because the mutation causes a less severe reduction in DNA-binding activity (Achermann et al., 2002).
5.2.2 Dax1
Dax1 (dosage-sensitive sex reversal adrenal hypoplasia congenita (AHC) critical region on the X chromosome, NR0B1) is also a member of the nuclear receptor transcription family but possesses an atypical DNA-binding domain. It negatively regulates transcription and is thought to act as a core- pressor of other nuclear receptors, including SF-1, rather than directly bind- ing to DNA (Iyer & McCabe, 2004). Like SF-1, it is expressed in the hypothalamus, pituitary gland, adrenal glands, and gonads (Ikeda et al., 1996; Swain, Zanaria, Hacker, Lovell-Badge, & Camerino, 1996). In human adrenal development, Dax1 is expressed from the earliest stages of the adrenal primordium with an overlapping expression pattern with SF-1, although at a lower level (Hanley et al., 2001). By 52 dpc, both are expressed throughout the entire cortex, and this is maintained through ges- tation but with lower expression of both by 18 weeks. In mice, Dax1 expres- sion drops during gestation in males and is restricted to the ZG by 10 weeks of age but remains expressed throughout the cortex in females (Mukai et al., 2002).
Mutations in DAX1 in humans cause a form of AHC (Muscatelli et al., 1994; Zanaria et al., 1994) in which patients present with adrenal insufficiency of both glucocorticoids and mineralocorticoids and an adrenal cortex with poorly developed adult zones but retention of cytomegalic FZ cells. Mice in which exon 2 of Dax1 is deleted surprisingly display enhanced adrenal steroid output (Yu, Ito, Saunders, Camper, & Jameson, 1998) until the cortex starts to regress and cytomegalic cells appear as the animals age, suggesting that the mutation is hypermorphic in mice (Scheys, Heaton, & Hammer, 2011). However, it has been shown that Dax1 and Oct3/4 form a regulatory circuit in embryonic stem (ES) cells, perturbation of which can cause differentiation, and Dax1 has there- fore been proposed to be involved in the maintenance of self-renewal (Sun et al., 2009). Given the subcapsular expression of Dax1 in mice, at least in males, it has been suggested that loss of Dax1 causes uncontrolled differen- tiation of adrenal stem/progenitor cells and their exhaustion may take lon- ger in mice than humans, resulting in a delayed regression of the cortex (Scheys et al., 2011) (see later).
5.2.3 Cbx2
The mouse polycomb group protein Cbx2 (chromobox2 or M33) is required for adrenal and gonadal development. A mouse model in which the C-terminus of Cbx2 is lost displays gonadal developmental defects with
indistinct genital ridges observed at 11.5 dpc and marked gonadal hypoplasia at 13.5 dpc, along with male-to-female sex reversal (Katoh-Fukui et al., 1998). Analysis of these mutants also detected a degree of adrenal hypoplasia at 18.5 dpc (Katoh-Fukui et al., 2005). Interestingly, adrenal levels of Sf-1 were reduced by approximately 50%, indicating a possible role for Cbx2 in the regulation of Sf-1.
5.2.4 Insr/IGFR1
The IGF1 receptor (IGFR 1) is expressed in the human fetal adrenal gland, in both the DZ and the FZ (Shigematsu et al., 1989). IGF2 is expressed at high levels throughout the fetal adrenal gland, but IGF1 is restricted to the capsule (Mesiano, Coulter, & Jaffe, 1993; Mesiano, Mellon, & Jaffe, 1993).
Recently, it has been reported that a mouse model in which both the insulin receptor and Igf1r are constitutively deleted displays reduced growth, gonadal dysplasia with male-to-female sex reversal, and adrenal develop- mental defects ranging from severe hypoplasia to agenesis (Pitetti et al., 2013). Signaling through these receptors appears to impact specifically on the AGP with an approximately 40% reduction in the number of SF-1-positive progenitor cells in the genital ridge at 11.5 dpc and a greater overall decrease in Sf-1 transcript levels, whereas other gross markers of development appeared normal, apart from a reduction in fetal size. The more deleterious effect on the adrenal gland than the gonads may result from this reduction in SF-1 expression (see later), and it is hypothesized that sig- naling through these receptors may upregulate SF-1 expression or phos- phorylate SF-1 and enhance its transcriptional activity.
5.3. Adrenal primordium development
5.3.1 Cited2
Cited2 (CBP/p300-interacting transactivator with ED-rich tail 2) is a tran- scriptional cofactor that binds with high affinity to p300 and CBP transcrip- tional coactivators. It can both positively and negatively regulate transcription: it not only inhibits the binding of CBP/p300 to HIF-1a (Bhattacharya et al., 1999), thus inhibiting transcriptional responses to hyp- oxia, but also mediates CBP/p300 binding to AP-2 (TFAP2) to activate transcription of AP-2-responsive genes (Bamforth et al., 2001; Braganca et al., 2003). It positively regulates transcription by promoting interaction of CBP/p300 with transcription factors such as AP-2 (Braganca et al., 2003), Lhx2 (Glenn & Maurer, 1999), and PPARa (Tien, Davis, & Vanden Heuvel, 2004). Cited2 is expressed in the coelomic epithelium
and adjacent mesenchyme of the urogenital ridge at 10 dpc and remains expressed at high levels in the adrenal cortex but at much lower levels in the gonads from 13.5 dpc (Val et al., 2007). Deletion of Cited2 in mice is embryonically lethal (Bamforth et al., 2001; Val et al., 2007) with embryos exhibiting markedly reduced adrenal development by 12 dpc (Val et al., 2007) and adrenal agenesis by 17.5 dpc (Bamforth et al., 2001), as well as cardiac, neural tube (Bamforth et al., 2001), and left-right patterning (Bamforth et al., 2004) defects. Expression of some gonadal markers is reduced at 11.5 dpc, suggesting an early delay in development, but this recovers by 13.5 dpc and gonadal development proceeds grossly normally as judged by these transcriptional patterns. However, further inspection reveals disrupted testis morphology at all further stages and delayed ovarian differentiation (Combes et al., 2010).
5.3.2 Wnt/B-catenin signaling
B-Catenin is a bifunctional protein; the majority regulates the interaction of cadherin complexes with the cytoskeleton in adherens junctions, but a cyto- plasmic pool responds to canonical Wnt signaling to act as a coactivator for transcription factors of the TCF/LEF. In the absence of Wnt signaling, cyto- plasmic ß-catenin is degraded by a complex containing GSK3B that phos- phorylates ß-catenin and targets it for degradation by the proteasome. Wnt signaling inhibits this degradation and allows nuclear accumulation of ß-catenin (for reviews, see Berthon, Martinez, Bertherat, & Val, 2012; El Wakil & Lalli, 2011). This and/or the expression of ß-catenin/TCF/ LEF-responsive genes allows the detection of cells transducing canonical Wnt signals. Transcriptionally active ß-catenin has been detected in the coe- lomic epithelium of the AGP and the mesonephros at 11.5 dpc (Usongo & Farookhi, 2012); by 12.5 dpc, it can be seen in some cells at the periphery of the adrenal primordium (Kim et al., 2008); and by 18.5 dpc, it localizes to subcapsular cell clusters where it remains expressed in adulthood. Subcapsu- lar clusters of strongly ß-catenin-expressing cells were identified immunohistochemically by 14.5 dpc (Kim et al., 2008). ß-Catenin is crucial for embryogenesis and deletion causes early embryonic lethality. Deletion of ß-catenin in mice in steroidogenic cells using an SF-1 cre driver demon- strated that at 12.5 dpc, the adrenal primordium is detectable and of approx- imately normal size. However, there are fewer SF-1-expressing cells and the levels of expression of the SF-1 target genes CYP11a1 and 3ßHSD are reduced. By 13.5 dpc, these, along with CYP21, which is unaffected at 12.5 dpc, are virtually undetectable, and cell proliferation is greatly reduced.
The ß-catenin-null adrenal glands are markedly smaller than wild-type adre- nal glands at 14.5 dpc and also contain many fewer SF-1-positive cells. By 16.5 dpc, the vast majority of the cells in the adrenal gland are TH-positive with almost no detectable SF-1-positive cells, indicating that the majority of the gland consists of chromaffin cells. By 18.5 dpc, the gland is no longer detectable.
5.4. Transcriptional control of SF-1
As discussed earlier, SF-1 is the major driver of adrenal development and function, and it has been demonstrated that forced expression of SF-1 in mouse ES cells (Crawford, Sadovsky, & Milbrandt, 1997) or bone marrow cells (Gondo et al., 2004) causes their differentiation into steroidogenic cells that can produce deoxycorticosterone and 17OH progesterone in response to ACTH stimulation. Furthermore, the expression of SF-1 under the con- trol of its fetal adrenal enhancer (FAdE) in transgenic mice (see later) results in the formation of larger adrenal glands and ectopic adrenal tissue, indicat- ing that it has caused nonadrenal lineage cells to switch cell fate (Zubair, Oka, Parker, & Morohashi, 2009). Investigation of the transcriptional con- trol of SF-1 during development has demonstrated that many of the defects described earlier can be viewed in the light of their role in activating Sf-1 expression or modulating SF-1-dependent transcription. Both Odd1 (Wang et al., 2005) and Sall1 (Nishinakamura et al., 2001) deletion cause reduced expression of Wt1. Wt1 binds to sites within the SF-1 promoter and together with the coactivator Cited2 drives the high levels of SF-1 expression required for adrenal primordium development (Val et al., 2007). SF-1 upregulates Dax1, which in turn represses SF-1 transcriptional activity and, hence, steroidogenesis (Clipsham & McCabe, 2003). It is not clear how disruption of SF-1 and its negative regulator can cause similar defects, but perhaps, it indicates a very fine control of SF-1 activity is required for adrenal development. However, Dax1 can function as a coactivator for SF-1 transcriptional activity in steroidogenic cells when expressed at high levels, and perhaps, the observations in vivo are complicated by the pleiotropic effects of these factors and not a simple consequence of their coactivity (Xu et al., 2009). SF-1 and ß-catenin cooperate to transcrip- tionally activate Dax 1 expression (Mizusaki et al., 2003) and the expression of other genes (Gummow, Winnay, & Hammer, 2003). Wnt4 expression is reduced in Sall1- (Nishinakamura et al., 2001) and Wt1-null mice, and it can upregulate Dax1 (Mizusaki et al., 2003), further demonstrating the
complexity of the transcriptional network specifying adrenal development (Essafi et al., 2011).
6. SIGNALING PATHWAYS INVOLVED IN ADRENAL DEVELOPMENT
6.1. Shh signaling
Sonic hedgehog (Shh) is member of the vertebrate hedgehog family of secreted ligands, along with Desert hedgehog (Dhh) and Indian hedgehog (Ihh); these perform a multitude of crucial roles during embryonic develop- ment and are also required in the adult for tissue maintenance, differentia- tion, and the regulation of stem cell populations (for reviews, see King, Guasti, & Laufer, 2008; Laufer et al., 2012). Secreted Hh ligands bind to the 12-pass transmembrane protein Patched-1 (Ptch1) expressed on Hh signal-receiving cells (Stone et al., 1996). In the absence of ligand binding, Ptch1 inhibits the actions of the 7-transmembrane G protein-coupled recep- tor Smoothened (Smo) by a poorly characterized mechanism, and members of the zinc finger transcription factor family Gli3 and Gli2 are proteolytically processed, losing their C-terminal activation domain (Pan, Bai, Joyner, & Wang, 2006). In this form, these transcription factors act to repress transcrip- tion, although the majority of Gli2 is degraded by the proteasome. The binding of Hh to Ptch1 relieves the inhibition it exerts on Smo and allows it to prevent the processing of the Gli transcription factors. Full-length Gli3 and Gli2 act as transcriptional activators. Gli1, which only acts as a transcrip- tional activator, is not expressed in the absence of Hh, but is upregulated by the pathway, and so can be used as a marker for active Hh signaling (Vokes et al., 2007).
Shh, but not Dhh or Ihh, is expressed in the adrenal primordium follow- ing separation from the AGP (Bitgood & McMahon, 1995; Boulkroun et al., 2011; Ching & Vilain, 2009; Guasti et al., 2011; Huang et al., 2010; King et al., 2009; Paul & Laufer, 2011) in cells just beneath the capsule. Its expres- sion remains restricted to this subcapsular cell population throughout gestation and adulthood. In rodents, Shh is expressed in relatively undifferentiated steroidogenic cells that express neither CYP11B1 nor CYP11B2 (Guasti et al., 2011; King et al., 2009). These cells are arranged in clusters interspersed between clusters of CYP11B2-expressing cells in mice (King et al., 2009), but in rats, the cells are in a continuous layer between the CYP11B2 and the CYP11B1 layers and separated from the CYP11B1 layer by the inner ZU (Guasti et al., 2011; Mitani et al., 1999)
(see later). In humans, SHH-expressing cells are also observed in subcapsular clusters (Boulkroun et al., 2011). Although costaining with SHH has not been performed in humans, CYP11B2 is detected in clusters of cells (Boulkroun et al., 2011; Nishimoto et al., 2010), and by analogy with the mouse adrenal gland, it is likely that the SHH-expressing cells are between these clusters. There is also a layer of cells between the CYP11B2- and the CYP11B1-expressing populations that may be analogous to the ZU (Nishimoto et al., 2010), suggesting that SHH expression in the human adrenal gland bears features of both the rat and the mouse (Fig. 7.1).
Holoprosencephaly is a consequence of the inactivation of the Hh pathway, with defects observed in SHH (Nanni et al., 1999), PTCH1 (Ming et al., 2002), and GLI2 (Roessler et al., 2003), and is often associated with adrenal hypoplasia (Dubourg et al., 2007). Smith-Lemli-Opitz syndrome (SLOS), a condition in which defects in 7-dehydrocholesterol reductase, an enzyme required for the formation of cholesterol from 7-dehydrocholesterol, is associated with abnormalities that overlap those seen in cases of impaired Hh signaling (Kelley & Hennekam, 2000), includ- ing holoprosencephaly. SLOS is also associated with adrenal insufficiency (Chemaitilly et al., 2003). This may be due to the requirement of cholesterol as the substrate for steroidogenesis and consequently adrenal function or may be linked to two observations regarding Hh signaling. First, Hhs are cova- lently linked to cholesterol, the only such proteins known to undergo this modification, which is critical for their signaling (Riobo, 2012), and second, 7-dehydrocholesterol ((pro-) vitamin D3), which accumulates in the plasma of SLOS patients (Bijlsma et al., 2006), can be transported out of the cell by Ptch1 and act as a negative regulator of Smo. Hypoadrenalism was also reported in the first description of Pallister-Hall syndrome (Hall et al., 1980), which is caused by a protein-truncating mutation in GLI3 (Hall et al., 1980). The introduction of a similar Gli3 mutation, which creates a constitutive repressor form of Gli3, into mice was reported to result in adre- nal agenesis (Bose, Grotewold, & Ruther, 2002), but this has recently been called into question (Laufer et al., 2012).
Homozygous deletion of Shh in mice is embryonically lethal, demon- strating the importance of this morphogen in development. Analysis of the adrenal gland at 14.5 and 16.5 dpc in Shh- - embryos, however, indi- cates that the adrenal primordium forms but is much smaller than in the wild-type (Ching & Vilain, 2009; Huang et al., 2010; King et al., 2009). Conditional homozygous deletion of Shh from steroidogenic tissues by crossing floxed Shh mice with Sf-1 cre mice is compatible with life, and these
animals have small adrenal glands both during embryogenesis and in the adult but with grossly normal histological appearance and zonation with a normal medulla (Ching & Vilain, 2009; Huang et al., 2010; King et al., 2009).
6.2. FGF signaling
Fibroblast growth factor (FGF) signaling controls early developmental pro- cesses such as cell movement during gastrulation, mesodermal and neuro- ectoderm formation, anterior/posterior patterning, and organogenesis (Ornitz & Itoh, 2001; Turner & Grose, 2010). FGFs are a large family of secreted glycoproteins that bind to four signaling FGF receptors, FGFR 1-4. The FGFRs are receptor tyrosine kinases with three extracellular immuno- globulin (Ig) domains, a single transmembrane domain, and an intracellular tyrosine kinase domain. FGFRs 1-3 are alternatively spliced in the third Ig domain such that an invariant exon IIIa is spliced to either exon IIIb or IIIc to produce IIIb or IIIc isoforms, which exhibit preferential binding to dif- ferent FGFs (Turner & Grose, 2010). Binding of FGFs to their cognate receptors activates a variety of different pathways in different settings: most commonly, Ras/MAPK activation controls cell proliferation and differen- tiation, Akt activation is associated with cell survival, and PKC activation is associated with cell migration (for reviews, see Bottcher & Niehrs, 2005; Dorey & Amaya, 2010).
Analysis of FGF and FGFR expression in the developing mouse adre- nal gland at 15.5 dpc detected FGFs 1, 2, and 9 and FGFRs 1 IIIc, 2 IIIb, 2 IIIc, and 3 IIIc in either predominantly the capsule (FGF2, 9) or the cortex (FGFR2 IIIb, IIIc, and FGFR3 IIIc) or both (FGFR1 IIIc), with FGFR2 IIIb and IIIc being expressed in subcapsular clusters of cells similar to Shh and ß-catenin (Guasti, Sze, Mckay, Grose, & King, 2013). FGF2 acts as a mitogen for adrenocortical cells both in culture and in gland regeneration experiments (Chu, Ho, & Dunn, 2009; Crickard, Ill, & Jaffe, 1981; Gospodarowicz, Ill, Hornsby, & Gill, 1977; Lepique et al., 2004) and has been shown to bind specifically to cells from the ZG (Basile & Holzwarth, 1994). The principal receptor for FGF2 is FGFR2 IIIc, and therefore, this observation is in line with the receptor expression analyses.
Embryos with a global Fgfr2 IIIb deletion have hypoplastic adrenal glands (Guasti et al., 2013; Revest et al., 2001), and deletion of both isoforms of FGFR2 from steroidogenic tissue recapitulates this phenotype and causes
male-to-female sex reversal (Kim et al., 2007), implying that FGFR2 is not necessary for AGP formation but is required for the subsequent growth and development of the adrenal gland.
6.3. Fetal adrenal growth
Anencephaly is a common congenital abnormality caused by the failure of the neural tube to close. The severity of the defect is variable but generally results in the absence of the hypothalamus, although the anterior pituitary gland is often spared (Swaab, Boer, & Visser, 1978). Often, these fetuses exhibit adrenal hypoplasia, which can be detected from between 13 and 18 wpc (Benirschke, 1956; Gray & Abramovich, 1980; Mazzitelli, Vauthay, Grandi, Fuksman, & Rittler, 2002), indicating that early formation of the adrenal gland proceeds normally in the absence of hypothalamic input. The hypoplasia is mainly observed in the FZ, with a relatively normally sized DZ and medulla (Bocian-Sobkowska, Malendowicz, & Wozniak, 1997) and normal cellular proliferation (Staton, Grilliot, & Parker, 2004), and pro- duction of the FZ-derived steroid hormone DHEA is reduced in these fetuses (Easterling, Simmer, Dignam, Frankland, & Naftolin, 1966). Fetal ACTH is detectable by 50 dpc in adrenal development (Goto et al., 2006) and is thought to be responsible for androgen secretion from the FZ (Carr, Ohashi, MacDonald, & Simpson, 1981) and also for the short window of cortisol production from the DZ at this time (Goto et al., 2006). Anencephalic pituitary glands show a reduction in ACTH produc- tion by midgestation, indicating a requirement for continued input from the hypothalamus (Begeot, Dubois, & Dubois, 1977; Osamura, 1977), and this is supported by the observation that administration of ACTH to anencephalic fetuses in utero restores the size of the FZ (Honnebier & Swaab, 1973). Based on these data, it has been proposed that ACTH, or pos- sibly another POMC peptide (Estivariz, Iturriza, McLean, Hope, & Lowry, 1982), is the primary regulator of adrenal development (Ishimoto & Jaffe, 2011). However, Pomc-null mice (Karpac et al., 2005) and Mc2r-null mice (Chida et al., 2007) have normal adrenal glands at birth, indicating that nei- ther ACTH nor any other POMC peptide is required for fetal adrenal devel- opment in this species. It is possible that because the rodent placenta, unlike humans, expresses CYP17 and is able to make estrogen (Arensburg, Payne, & Orly, 1999; Durkee et al., 1992), whereas this is formed from adre- nal androgens secreted from the human FZ under the control of ACTH, there is no need for ACTH signaling in utero in mice. This would indicate
that the role of ACTH in humans is to maintain pregnancy through its actions on steroidogenesis rather than via its effects on growth.
It has also been hypothesized that ACTH itself is not mitogenic, but upregulates the expression of other growth factors such as IGF2 (Voutilainen & Miller, 1987) and FGF2 (Mesiano, Mellon, Gospodarowicz, Di Blasio, & Jaffe, 1991). Both these factors are potent mitogens for fetal adrenal cells in vitro (Crickard et al., 1981; Mesiano, Coulter, & Jaffe, 1993; Mesiano, Mellon, & Jaffe, 1993). IGF2 is not expressed in the adult cortex, but is proposed to be a major regulator of fetal adrenal growth (for review, see Mesiano & Jaffe, 1997). FGF2 has a more profound effect on the growth of the DZ than the FZ (Crickard et al., 1981).
6.4. Fetal zone involution and X zone regression
FZ involution takes place rapidly during the first 2 weeks following birth and is known to involve apoptosis of cells in the inner FZ (Jirasek, 1980; Spencer et al., 1999). Activin and inhibin are expressed in the FZ (Spencer, Rabinovici, Mesiano, Goldsmith, & Jaffe, 1992), and in vitro studies demon- strate that activin can inhibit FZ cell proliferation by triggering apoptosis, with no effect on DZ cells, indicating a possible role in involution (Spencer et al., 1999). The X zone of the mouse adrenal gland expresses high levels of activin receptors and Smad2, a downstream effector in the activin pathway, and activin injection into intact mouse adrenal glands ex vivo, or in vitro treatment of cell cultures, caused apoptosis specifically in the X zone cells (Beuschlein et al., 2003). These data indicate a potential equiv- alence of the FZ and X zones (see later).
X 7. MODELS OF DEVELOPMENT
7.1. SF-1 FAdE
As discussed previously, the primary driver of the development of the AGP appears to be SF-1. It is the first identified marker of these anlagen, and in its absence, the primordium undergoes apoptosis after specification. Analysis of the transcriptional regulation of SF-1 in the adrenal primordium revealed the presence of an adrenal gland-specific enhancer, referred to as the FAdE. The FAdE is active in mouse development from 10.5 dpc, the time at which the adrenal primordia and gonadal primordia separate, and in all cells of the adrenal primordium until 13.5 dpc. By 15.5 dpc, its activity was restricted to the inner cortex around the medulla and persisted in the X zone of the adult
mouse until its regression (Zubair et al., 2006; Zubair, Parker, & Morohashi, 2008). This enhancer, which is located in the fourth intron (Zubair et al., 2006) and is conserved in mammals (Gardiner, Shima, Morohashi, & Swain, 2012), contains binding sites for Pbx complexes as well as SF-1 itself. Mutation of the Pbx binding sites in FAdE abolishes the expression of SF-1 in the adrenal primordium in transgenic mice (Zubair et al., 2006), and fur- thermore, as described earlier, Pbx1 is expressed early in the development of the urogenital ridge and its absence causes greatly reduced SF-1 expression in Pbx1-null mice (Schnabel et al., 2003). Mutation of the SF-1 sites does not impair the activity of the enhancer at 11.5 dpc, but by 17.5 dpc, activity was abolished (Zubair et al., 2006), suggesting that Pbx1 is required for initiation of SF-1 expression in the adrenal anlagen but its prolonged expression requires SF-1 to act in a feedforward autoregulatory mechanism.
The FAdE transgene was used in lineage-tracing experiments to drive expression of tamoxifen-inducible cre recombinase. If pregnant mothers were injected with tamoxifen at 10.5 dpc, the entire cortex was labeled at 14.5 dpc, but when injected at 12.5 and 13.5 dpc, there was a progressive decrease in labeling in cells of the outer cortex, and injecting at 14.5 dpc did not label the outer cortical cells at 15.5 dpc (Zubair et al., 2008). Inter- estingly, injecting at 10.5 dpc also revealed some weak labeling of the gonads, but this was not seen at later time points, suggesting that FAdE is repressed in the gonadal primordium. When similar experiments were per- formed but the adrenal glands were not studied until the animals were 2 months of age, those injected at 11.5 dpc were still labeled across the cor- tex, but those injected at 14.5 dpc retained no label at all (Zubair et al., 2008). This study demonstrates that mice adrenal glands possess fetal and DZs equivalent to those in the human adrenal gland and that the founder population of fetal adrenal cells can give rise to the DZ in early development of the cortex but loses this potential from around 14.5 dpc. Persistent label- ing of the X zone by the FAdE transgene also demonstrates that the original FZ becomes the X zone.
These observations argue against earlier ideas that the primordium dif- ferentiates into the FZ while the DZ is formed from another group of migrating coelomic epithelial cells (Crowder, 1957; Uotila, 1940) or alter- natively that only the inner portion of the primordium differentiates to form the FZ and the outer portion retains its primordial phenotype (Jirasek & Lojda, 1964). It is not known whether such a mechanism takes place in humans, but the very high conservation of the FAdE sequence across species would argue for its role in human adrenal development.
7.2. Shh/Gli1 lineages
An alternative view of adrenal development was provided by studies on Shh signaling in the adrenal cortex. Shh expression has been first detected at around 12.5 dpc in mouse adrenal glands; however, more sensitive lineage analyses using an Shh cre driver detected Shh expression as early as 11.5 dpc, in the adrenal glands but not in the gonads, suggesting that Shh expression is activated exclusively in cells of the adrenal primordium almost immediately after its separation from the AGP (King et al., 2009; Laufer et al., 2012). In this analysis, the number of labeled cells in the adrenal increased rapidly to 13% of all SF-1-positive cells by e12.5, mainly in the subcapsular region, and to 75% by 13.5 dpc, spread throughout the developing cortex. Analysis of the adrenal glands from these experiments shortly after birth showed that all cortical cells were labeled, including those of the ZG, ZF, and X zone (King et al., 2009), but not the capsule or medulla. When similar experi- ments were performed with an inducible Shh cre-T2 driver, labeled cells were seen at the periphery of the gland at birth following tamoxifen injec- tion at 14.5 dpc, and after 28 days, columns were observed, which almost spanned the cortex to the medulla boundary. Adult mice were also injected and similar results were obtained with 7- and 13-day chases, with the labeled cells coexpressing CYP11B2 or CYP11B1 in increasing numbers. These data indicate that Shh-expressing cells are candidate stem/progenitor cells both in the fetal adrenal gland and for adult maintenance of the gland.
Deletion of Shh from the cortex causes adrenal hypoplasia but it does not act upon cortical cells; rather, it signals to mesenchymal cells in the capsule and in the periphery of the cortex. These are identified by their expression of Hh-responsive genes, such as Gli1, Ptch 1, and FoxD2, and do not express the pan-steroidogenic markers SF-1 and CYP11A1. The lack of requirement for Hh signaling to cortical cells is evidenced by the fact that deletion of Smo from cortical cells using the SF-1 cre driver has no effect on adrenal development (King et al., 2009). However, the adrenal hypoplasia was asso- ciated with a much thinner capsule with a greatly reduced proliferative capacity (Huang et al., 2010; King et al., 2009), suggesting that the effect of Shh was therefore chiefly via the capsule. Lineage-tracing experiments using inducible Gli1 cre drivers demonstrated that the capsule and subcap- sular mesenchymal cells were initially labeled by injection of tamoxifen at 14.5 or 15.5 dpc, but clusters of labeled cells, which expressed steroidogenic markers (King et al., 2009), were observed in the cortex (Huang et al., 2010; King et al., 2009) and expanded into columns with extended chases. Similar
to the tracing using Shh, these columns eventually spanned the cortex to the medulla (Huang et al., 2010; King et al., 2009). At least some of these labeled cells were observed to express Shh (King et al., 2009). Postnatal induction of the Gli cre driver also labeled columns of cells entering the cortex (King et al., 2009). Taken together, these lineage-tracing studies provide direct confirmation of the centripetal migration hypothesis and that the stem/pro- genitor cell populations reside at the periphery of the gland. They imply that Shh signals to a mesenchymal population of cells that differentiate from a nonsteroidogenic to a steroidogenic phenotype, at least in part via an Shh-expressing intermediate (Fig. 7.4). These data also imply that the defin- itive cortex develops from the capsule and the adrenal cortex and is of both coelomic epithelial and mesenchymal origin and are therefore contradictory to the results from the FAdE experiment, which suggests that the cortex is entirely of SF-1-expressing coelomic epithelium origin.
One explanation for this conundrum has been to invoke a mechanism in which cells in the newly separated adrenal primordium enter the forming adrenal capsule whereupon their SF-1 expression is immediately extinguished before the onset of Shh expression. SF-1 expression is subse- quently reactivated upon entry of these cells back into the cortex (Wood & Hammer, 2011). One candidate for a factor repressing SF-1 in the newly forming adrenal capsule is the helix-loop-helix transcription factor Pod1. Loss of Pod1 in mice leads to the expansion of the SF-1-expressing domain in the testes, with increased differentiation of the steroidogenic Leydig cells and ectopic expression of SF-1 and SF-1 target genes (Cui et al., 2004). Pod1 can repress SF-1 expression through inhibiting binding of USF to an E box in its promoter (Cui et al., 2004). Pod1 is exclusively expressed in the capsule of the adrenal gland in adult mice (Kim & Hammer, 2007) and analysis of adrenal glands from Pod1 null mice also demonstrated ectopic expression of SF-1 in some regions of the capsule (Kim & Hammer, 2007). It has there- fore been hypothesized that Pod1 downregulates Sf-1 expression in the fetal adrenal cells upon recruitment into the capsule. While this is an attractive hypothesis, other data do not support it. An SF-1 BAC transgene driving cre does not label the capsule (King et al., 2009) and the FAdE lineage trac- ing does not appear to label it either (Zubair et al., 2008). Furthermore, Pod1 expression in the developing adrenal gland is apparently detected within the cortex but not in the capsule at 16.5 dpc (Maezawa et al., 2012), at a time when SF-1 is undetectable in the capsule. The resolution of these apparently conflicting studies awaits further investigation.
10.5 dpc FAdE active
11.5 dpc FAdE active
14.5 dpc FAdE inactive
16 dpc FAdE inactive
7.3. Stem/progenitor cells
The Shh/ Gli1 lineage-tracing studies referred to earlier provide genetic evi- dence for the long-held view that adrenocortical stem/progenitor cells are located in the capsule and subcapsular region of the gland. Analysis of
proliferation and apoptosis in Shh mutant adrenal cortices revealed a reduc- tion in PCNA staining in mutants compared to wild-type adrenal glands from 13.5 dpc that persisted through to the adult (Ching & Vilain, 2009), but no apparent increase in apoptosis (Ching & Vilain, 2009; Huang et al., 2010). These data suggest that Shh signaling to the capsule is not required for the production of a cortical survival signal from this compart- ment but may produce a proliferative signal. Interestingly, Huang et al. observed that the greatest effect on cell proliferation in Shh-null adrenal glands was observed in the capsule, indicating that Shh may act as a mitogen for the capsule (Huang et al., 2010). The remarkably thin capsule in these mutants may result from the lack of trophic support from the Shh signal, or Shh may alternatively be acting as a chemoattractant to promote the coa- lescence of the surrounding mesenchyme to form the capsule, or it may reg- ulate the differentiation of the capsule into the steroidogenic lineages, with rapid depletion from the capsule during development as a consequence of its absence.
Thus, there is an Shh signal from the cortex to the capsule and potentially an Shh-dependent proliferative signal acting on the cortex from the capsule. Candidates for these signals include FGF2, exclusively expressed in the capsule of 15.5 dpc mouse adrenal glands and known to be a mitogen for adrenocor- tical cells, at least in vitro (see earlier), and Wnts. Deletion of B-catenin is extremely deleterious for adrenocortical cell proliferation, with an approxi- mately 75% decrease in BrdU uptake in cells at 13.5 dpc (Kim et al., 2008), but the identity of the Wnts acting upon it is yet to be determined. Wnt4 is a candidate but is principally produced by cortical cells. Wnts exclu- sively expressed in the capsule of the rat adrenal gland include 2, 6, and 8b (Cavlan D and King P, unpublished results) and may represent Shh-dependent proliferative signals. It is as yet not known whether Shh signaling is required for FGF or canonical Wnt signaling in the subcapsular region. Interestingly, Fgfr2 IIIb-null mice have thicker capsules that display a disorganized arrange- ment of cells rather than the normal stratified arrangement, a phenotype also observed in Mc2r-null mice (Chida et al., 2007). Potentially, this indicates that signaling through FGFR2 IIIb or the MC2R regulates capsule growth, per- haps through regulation of the Shh signal in the subcapsular cells. This has not been investigated for Mc2r-mutant adrenal glands, but Fgfr2 IIIb mutant cap- sules have enhanced cell proliferation as well as upregulated Gli1 expression, suggesting that FGF signaling may in fact be a negative regulator of Shh expression. Further examination of the reciprocal signaling between the cap- sule and the subcapsular progenitor cells is warranted.
8. CONTROL OF POSTNATAL GROWTH, ZONATION, AND REMODELING
The adrenal cortex is a highly dynamic organ: there being a constant centripetal migration of cells during under normal conditions, the cortex rapidly responds to requirements for hormonal production by altering the relative sizes of the zones in response to alterations of HPA or RAS activity in a process known as adrenal remodeling. Furthermore, like the liver, the adrenal has the ability to regrow following injury. The stem/progenitor populations identified earlier are likely to be involved in these hypertrophic and hyperplastic processes and in the control of zonation, but genetic ana- lyses have yet to be undertaken to follow the lineages recruited in these processes.
8.1. Normal growth
ACTH is often considered the principal regulator of adrenal growth. Under normal conditions, cell proliferation in the adrenal cortex displays a circa- dian rhythm with a peak observed at 4 am, principally in the outer ZF. This circadian rhythm follows that of ACTH, suggesting that this is the main stimulator of proliferation (Mitani et al., 2003; Miyamoto, Mitani, Mukai, Suematsu, & Ishimura, 1999). Furthermore, it is well established that hypophysectomy of experimental animals results in adrenocortical atro- phy (Estivariz, Carino, Lowry, & Jackson, 1988; Estivariz, Morano, Carino, Jackson, & Lowry, 1988), while excess ACTH states such as Cushing’s dis- ease cause adrenocortical hyperplasia (Bertagna, Guignat, Groussin, & Bertherat, 2009). As discussed previously, Pomc-null mice are born with normal adrenal glands (Karpac et al., 2005), but it has been found that soon after birth, the gland atrophies, indicating a requirement for POMC pep- tides for adrenal maintenance and growth (Coll et al., 2004; Yaswen, Diehl, Brennan, & Hochgeschwender, 1999). Administration of ACTH restored the adrenal weight and function in these animals (Coll et al., 2004, 2006), but ACTH replacement does not restore adrenal size in hypophysectomized rats (Payet & Lehoux, 1980). Furthermore, ACTH has been shown to be antimitogenic in primary adrenal cells and mouse Y1 adrenocortical cells (Masui & Garren, 1971; Rocha, Forti, Lepique, & Armelin, 2003).
8.2. Zonation
The zonation of the adrenal cortex is established perinatally with the onset of CYP11B2 expression in the ZG in humans and rodents. The juxtaposition of the proposed capsular or subcapsular stem/progenitor cells with the ZG and ZF raises some interesting questions regarding the differentiation pro- cesses that occur during centripetal migration. In mice, where ZG and ZF cells are in direct contact, centripetal migration would predict the ability of ZG cells to transdifferentiate into ZF cells. However, in rats, these two populations are separated by the ZU (Mitani et al., 2003). This zone has been further characterized and shown to be subdivided in to two other zones, an outer Shh-expressing zone, named the outer ZU (oZU), and a smaller inner layer approximately one to three cells wide that does not express Shh, referred to as the inner ZU (iZU) (Guasti et al., 2011). This observation represents a conundrum for the maintenance of rat zonation. If, as in the mouse, the capsular cells delaminate and enter the cortex to become ste- roidogenic, then, at first glance, their initial identity would appear to be CYP11B2-positive ZG cells rather than Shh-positive cells. However, it is possible that the capsular cells first express Shh upon entering the cortex and these cells migrate through the CYP11B2-expressing cells to the oZU. Bidirectional differentiation of the Shh-positive progenitor cells of the oZU would then form the ZG and ZF, as has been proposed (Mitani et al., 1999, 2003). The iZU expresses high levels of steroidogenic enzymes, including CYP11A1, MC2R, and its accessory factor MRAP, and this might indicate that the iZU represents a differentiating TZ, which has lost Shh expression and is converting to a more steroidogenic phenotype, although it has yet to acquire CYP11B1 expression (Gorrigan, Guasti, King, Clark, & Chan, 2011). A pool of CYP11B1/CYP11B2-negative cells have been found in the human adrenal cortex (Nishimoto et al., 2010), suggesting the existence of the ZU in humans and the possibility of similar mechanisms of progenitor cell differentiation described for the rat.
The establishment of zonation in mice could also involve bidirectional differentiation of the Shh-expressing population. If this is not the case, and cells can transdifferentiate from a ZG to a ZF identity, the very sharp boundary between the ZG and the ZF must indicate that positional cues such as steep gradients of morphogens control these events. Trans- differentiation from ZG to ZF identity has not been formally demonstrated but is an interesting area for future study.
8.3. Remodeling
These considerations of stem/progenitor cell differentiation become all the more pertinent when remodeling is considered. Steroidal output responds rapidly to changes in HPA or RAAS activity, and when these are applied chronically in experimental models, profound changes in the size and ste- roidogenic expression of the zones are observed. For example, activation of the RAAS under low-sodium or high-potassium conditions causes a rapid expansion of the ZG to produce more CYP11B2-expressing cells and hence secrete more aldosterone, in rodent models (Romero et al., 2007), and adre- nal remodeling with increased CYP11B2 expression has been reported in adrenal glands from patients with primary aldosteronism (Boulkroun et al., 2010, 2011). Conversely, suppression of the RAAS under high- sodium conditions, or by the administration of angiotensin-converting enzyme (ACE) inhibitors, causes a rapid shrinking of the ZG with conse- quent reduction in CYP11B2 expression and reduction in blood volume (Brennan, Chittka, Barker, & Vinson, 2008; Pignatelli et al., 2000). Similar changes occur in the ZF; HPA axis activation by chronic ACTH adminis- tration expands the ZF to secrete and express more CYP11B1 and thus secrete more glucocorticoid, but suppression by dexamethasone causes its contraction (Dallman, 1984). The processes involved in remodeling may involve a combination of enhanced differentiation of stem/progenitor populations, dedifferentiation of mature steroidogenic cells, cell prolifera- tion, and apoptosis. For example, new ZG cells produced by RAAS activa- tion may differentiate from capsular and/or subcapsular stem/progenitor populations, or ZF cells may transdifferentiate to become ZG cells. Loss of ZG cells following RAAS inhibition may involve their dedifferentiation back into Shh-expressing cell populations or apoptosis (McEwan, Lindop, & Kenyon, 1996). Activation of the RAAS or HPA axis causes enhanced pro- liferation in the ZU and the zone being remodeled, the ZG and ZF, respec- tively (McEwan, Lindop, & Kenyon, 1995; McEwan et al., 1996). As yet, the mechanisms controlling these processes are unclear, although migration of BrdU labeled cells from the ZU into the ZG in the adrenal glands of rats following RAAS activation (Mitani et al., 2003).
8.4. Regeneration following surgical injury
8.4.1 Enucleation
These studies, in which the adrenal capsule is slit and the gland squashed to remove the inner ZF and medulla, demonstrate the remarkable regeneration
properties of the adrenal cortex that within 4 weeks can reestablish the ZG and ZF from the remaining capsule and adherent ZG cells (for review, see Bland, Desclozeaux, & Ingraham, 2003). This regeneration also occurs in humans (Feuerstein & Streeten, 1991). During adrenal regeneration in rats, CYP11B2 expression is lost in the remaining adherent ZG cells before an increase in proliferation in all cortical cells until CYP11B2 is reexpressed in the ZG, around 10 days postsurgery, and proliferation in this zone returns to normal levels. Proliferation remains increased throughout the ZF at this time point but returns to normal in the inner ZF by 28 days, remaining high in the outer ZF adjacent to the ZU (Ennen, Levay-Young, & Engeland, 2005). Shh or ß-catenin expression has not been documented during these experiments, but Dlk1 (Pref-1, ZOG), which is coexpressed with Shh in the rat ZU (Halder et al., 1998), is rapidly downregulated and not reexpressed until the cortex has regenerated, consistent with its role as a negative regu- lator of cell differentiation in other systems (Sul, 2009). Whether Shh and/or ß-catenin expression is similarly affected is not known at present. Interest- ingly, cell proliferation is observed in the adrenal capsule (Reiter & Pizzarello, 1966), which becomes initially thickened (Skelton, 1959), indi- cating a potential involvement of capsular cells in regeneration.
8.4.2 Unilateral adrenalectomy
Compensatory growth of the remaining adrenal gland following unilateral adrenalectomy is another paradigm of regeneration following injury. Unlike in adrenal enucleation, only growth of the ZF is observed following unilat- eral adrenalectomy to compensate for the loss of adrenal mass and resultant glucocorticoid deficit with proliferation being observed in the outer ZF (Engeland, Ennen, Elayaperumal, Durand, & Levay-Young, 2005). Cell proliferation is increased threefold in the outer ZF within 2 days of surgery, but, in contrast to enucleation, no increase in ZG proliferation is seen.
In all these remodeling paradigms, proliferating cells are located in and around the ZU, and this has led to the proposal that this is a stem cell zone in rats (Mitani et al., 1999, 2003). The expression of Shh in this region is highly suggestive of the involvement of this cell population in remodeling, and this merits further study. Interestingly, compensatory growth following adrenalectomy is not observed in Sf-1+ mice (Beuschlein et al., 2002).
8.5. ACTH and remodeling
Dax 1 is transcriptionally inhibited by ACTH and activated by glucocorti- coid (Gummow, Scheys, Cancelli, & Hammer, 2006; Ragazzon et al.,
2006), and given the inhibition Dax1 exerts over steroidogenesis, this potentially serves as a negative feedback loop to control glucocorticoid production in adrenocortical cells. However, as discussed earlier, Dax1 can function as a negative regulator of ES cell differentiation, and it is tempting to speculate, based on its subcapsular localization, at least in male mice, that it may play a similar role in controlling the mobilization of adre- nal stem cell populations under the control of the HPA axis (Kim & Hammer, 2007). However, the role for ACTH in regulating proliferation during remodeling is controversial. Clearly, ACTH administration can provoke hyperplasia in the ZF, and the expression of MC2R and MRAP in the iZU (Gorrigan et al., 2011) may suggest a role for ACTH in the differentiation of these cells towards the ZF phenotype in vivo. This may involve downregulation of Dax1 expression to allow differentiation of the progenitor cells to become ZF cells. Alternatively, dexamethasone, which blocks the HPA axis and lowers POMC production, may upregulate Dax1 expression and inhibit the formation ZF cells. However, the data from remodeling experiments are conflicting. Dexamethasone administra- tion has been shown to block ZF proliferation and compensatory growth following unilateral adrenalectomy in some studies (Engeland et al., 2005; Lowry, Silas, McLean, Linton, & Estivariz, 1983; Phillips, Crock, & Funder, 1985), but not others (Engeland, Shinsako, & Dallman, 1975; Grizzle & Dunlap, 1984). Hypophysectomy, the complete removal of the pituitary gland, blocks regeneration following enucleation (Estivariz, Carino, et al., 1988; Estivariz, Morano, et al., 1988) but does not completely block compensatory growth (Dallman, 1984; Dallman, Engeland, Holzwarth, & Scholz, 1980), and ACTH has even been shown to inhibit this process (Dallman et al., 1980). Nevertheless, hypophysec- tomy does reduce the overall extent of compensatory growth, suggesting the involvement of a pituitary factor. One such candidate for roles in both processes is N-POMC, which is derived from the secreted POMC cleav- age product pro-y-MSH. Neutralizing antibodies against pro-y-MSH inhibit both adrenal regeneration and compensatory growth, but pro-y- MSH has no mitogenic activity (Lowry et al., 1983). It was postulated that pro-y-MSH was cleaved at the adrenal gland to provide local sources of mitogenic N-POMC, and, subsequently, a protease expressed specifically in the adrenal cortex, AsP, principally at the outer ZF boundary in rat adre- nal glands, was isolated that cleaved pro-y-MSH to N-POMC and whose expression was significantly increased during compensatory growth (Bicknell et al., 2001; Estivariz, Carino, et al., 1988; Estivariz, Morano,
et al., 1988; Lowry et al., 1983). Interestingly, however, N-POMC cannot restore adrenal growth in Pomc-null mice (Coll et al., 2006).
These studies may suggest that ACTH is not involved in adrenal cell pro- liferation during adrenal remodeling, but it has been reported that the ele- vated ACTH levels that accompany enucleation are required for the dedifferentiation of the ZG cells that is an immediate response in the reg- enerating adrenal gland (Engeland & Levay-Young, 1999). Whether this works via inhibiting Dax1 expression has not been reported. ACTH does not affect proliferation in the ZG; the cell proliferation observed by placing animals on a low-sodium diet is inhibited by type 1 angiotensin receptor antagonists (McEwan et al., 1996), indicating that cell growth in this zone is controlled by the RAAS (Chatelain, Montel, Dickes-Coopman, Chatelain, & Deloof, 2003).
If N-POMC, rather than ACTH, provides the pituitary signal for com- pensatory growth, what is the nonpituitary signal observed? There is evi- dence for a neurally mediated signal consisting of afferent and efferent connections between the adrenal gland and the hypothalamus, with mechanical perturbation of the nerve-bearing adrenal pedicle causing increased proliferation in the contralateral adrenal gland (Dallman, Engeland, & Shinsako, 1976), and in support of this, compensatory growth is blocked by interruption of the nerve supply (Kleitman & Holzwarth, 1985). FGF2, recognized as a potent mitogen for adrenocortical cells in vitro, has been suggested to mediate this neural effect based on the obser- vation that suramin, which is thought to block FGF2 binding to its receptors in the adrenal cortex, inhibits compensatory growth, although there is little supporting data (Basile & Holzwarth, 1994).
X 9. DISEASES OF THE ADRENAL CORTEX
Several genetic mutations that result in adrenal agenesis or aplasia have been referred to in previous sections, and often, these are not compatible with life and therefore do not manifest as causes of adrenal insufficiency in children or adults. A number of adrenal insufficiency syndromes have been identified and elucidating their genetic causes will be important for fur- ther understanding mechanisms of adrenal development, growth, and main- tenance. These syndromes include those in which there is a relatively isolated defect in adrenal development; lack of trophic support from the pituitary gland; disorders of steroidogenesis; and occasionally, but less infor- matively, autoimmune destruction of adrenal tissue. Finally, adrenal cancer
will be discussed. For a more extensive appreciation of syndromes including adrenal insufficiency, see Else and Hammer (2005) and Ferraz-de-Souza and Achermann (2008).
9.1. IMAGe
AHC has been considered in terms of the role of SF-1 and Dax1. However, it also forms a feature of the rare IMAGe (intrauterine growth restriction, metaphyseal dysplasia, AHC, and genital anomalies) syndrome (Vilain et al., 1999). Most affected individuals are born small and develop skeletal abnormalities at the ends of the long bones, scoliosis, or osteoporosis later in life. Males present with genital malformations including micropenis and undescended testicles and urethra anomalies, but the most clinically important condition of the syndrome is adrenal insufficiency, which shortly after birth causes salt wasting, hypoglycemia, and shock due to loss of both mineralocorticoid and glucocorticoid synthesis and can be life-threatening. This syndrome is not associated with mutations in either SF-1 or Dax1, but recently, a candidate gene has been reported, CDKN1C, which is paternally imprinted and encodes the cell cycle regulator p57Kip2 (Arboleda et al., 2012). Mutations were detected in the PCNA-binding domain of p57Kip2 and interaction between the two proteins is disrupted. p57Kip2- null mice, however, display adrenal hyperplasia, reflecting its role as a cyclin-dependent kinase inhibitor (Pateras, Apostolopoulou, Niforou, Kotsinas, & Gorgoulis, 2009). p57Kip2 is expressed in the cortex with highest levels of expression adjacent to the capsule in both humans (Arboleda et al., 2012) and rats (Kobayashi et al., 2006). Its expression is upregulated by ACTH treatment in the ZG in rats, and it is tempting to speculate that this is to prevent proliferation in this zone. The discrepancy between these observations suggests that the loss of PCNA binding is insuf- ficient to create a loss-of-function mutation of p57Kip2, and further conse- quences of this mutation that causes gain of function in IMAGe, such as the potential impact upon its ubiquitination and stability (Arboleda et al., 2012), remain to be elucidated.
9.2. Familial glucocorticoid deficiency
In contrast with AHC, familial glucocorticoid deficiency (FGD) is a defect of glucocorticoid production with no effect on the production of mineral- ocorticoids from the ZG (Meimaridou et al., 2013). Patients typically pre- sent in infancy and childhood with a low cortisol and raised ACTH. Low
cortisol levels manifest in hypoglycemia, failure to thrive, and recurrent infections, which can be fatal if untreated by hormone replacement. Some- times, ACTH levels are high enough to cause hyperpigmentation via its actions on MC1R (Meimaridou et al., 2013). Several mechanisms have come to light over the recent years.
9.2.1 MC2R2
Mutations of the ACTH receptor MC2R were the first identified cause of FGD and account for approximately 25% of known cases. More than 40 have now been identified. Varied aspects of the receptor, from ACTH binding to signaling, are blocked by inactivating mutations targeting amino acids throughout the GPCR, prohibiting adrenal cortisol production in response to pituitary signals. Untreated patients have adrenal glands with severely atrophied ZF and ZR at postmortem (Clark & Weber, 1998).
9.2.2 MRAP
MRAP is a single transmembrane protein that binds MC2R via its trans- membrane domain and exports it from the endoplasmic reticulum to the plasma membrane (Metherell et al., 2005). Mutations in this protein account for a further 20% of FGD cases and include those that result in loss of the transmembrane domain and hence sequestering of the receptor in the ER and mutations that retain trafficking function but do not allow the formation of a signaling-competent receptor (Chung, Chan, Metherell, & Clark, 2010; Hughes et al., 2010). MRAP and MC2R expression patterns overlap in rat fetal and adult adrenal glands (Gorrigan et al., 2011), displaying a gradient of expression from a maximum in the ZU and fading to the inner ZF. The abil- ity of the ZU to respond to ACTH signals suggests that these cells are primed to convert to a ZF identity and may well be the cells that proliferate with a circadian rhythm discussed earlier.
9.2.3 StAR/CYP11A1
Almost half of all cases of FGD are caused by an inability to respond to ACTH. Further causes of FGD have been identified that implicate steroido- genesis, with mutations in StAR (Metherell et al., 2009) and CYP11A1 (Parajes et al., 2011; Sahakitrungruang, Tee, Blackett, & Miller, 2011). These are unexpected findings because mutations in these genes had previ- ously been found in another defect of steroidogenesis, lipoid congenital adrenal hyperplasia (LCAH), which, because of the central role of these genes in steroidogenesis, involves severe defects in glucocorticoid,
mineralocorticoid, and sex hormone synthesis, with accumulation of cholesterol in the cells causing cell death (King, Bhangoo, & Stocco, 2011). The mutations identified in FGD appear to have a less severe pheno- type than do the ones in LCAH and selectively affect steroidogenesis in the ZF. This is likely to be because the ZF cells have a more steroidogenic phenotype than ZG cells, being lipid-rich and containing mitochondria with tubulovesicular cristae. Mutations in these steroidogenic enzymes account for a further 5% of cases of FGD.
9.2.4 MCM4
An unusual form of FGD was observed in a cohort of Irish travelers in whom the condition developed relatively late in childhood following a period of normal function. The affected individuals also presented with chromosomal breakage, natural killer cell deficiency, and short stature (Gineau et al., 2012; Hughes et al., 2012). Genetic analysis of the cohort for a gene responsible for FGD or NK cell deficiency identified the same candidate, minichromosome maintenance-deficient 4 homologue (MCM4). This is a component of the heterohexameric prereplication complex along with MCM2, 3, 4, 6, and 7 that forms at the origin of replication to initiate DNA synthesis during cell division. The identified mutation was a missense mutation in the splice acceptor site of exon 2, which was predicted to lead to a one nucleotide shift in this site, thus generating a frameshift that caused a prematurely terminated protein consisting of only 27 amino acids. Given the essential role of this factor in cells, as evidenced by the fact that Mcm4-null mice that died before implantation (Shima et al., 2007), and the serious foreshortening of the pro- tein predicted by the mutation identified, it was very surprising that this homozygous mutation was compatible with life. However, although full- length MCM4 could not be detected in lymphocytes from affected individ- uals, shorter proteins lacking the N-terminus were detected, potentially being translated from in-frame ATGs located in exon 2. The N-terminal 130 amino acids are dispensable for activity of the protein (Masai et al., 2006), and the mutant protein appears to be able to retain function in the majority of tissues in these patients. It is unclear why the adrenal gland should be particularly affected by this mutation, but the study of the adrenal glands in a mouse model that reduces MCM4 to the lowest levels compatible with life demonstrated that their steroidogenic cells are increasingly replaced by nonsteroidogenic spindle-shaped cells as the animals age, suggesting a pro- gressive loss of steroidogenic capacity in keeping with the FGD phenotype (Hughes et al., 2012).
9.2.5 NNT
The most recent causative mutations reported for FGD have been found in nicotinamide nucleotide transhydrogenase (NNT) (Meimaridou et al., 2012). NNT is located in the inner mitochondrial membrane where it reduces NADP+ to NADPH, which is in turn used to maintain high reduced glutathione levels. This allows glutathione peroxidases to detoxify harmful reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, which are produced during oxidative phosphorylation and ste- roidogenesis. Twenty-one mutations have been found spread throughout the gene in 15 families and knockdown of NNT levels in the human adre- nocortical carcinoma cell line H295R lowered reduced glutathione levels as well as increasing levels of ROS and activating apoptosis. Comparison of the adrenal glands from substrains of C57Bl6/J, one of which contains a spon- taneously occurring Nnt mutation, showed that although the appearance of the glands and the steroidogenic gene expression is similar, increased apo- ptosis was observed in the ZF and corticosterone levels were reduced both basally and upon ACTH stimulation in the Nnt mutant substrain, indicating an impairment of steroidogenesis. As ROS are produced as a by-product of cellular metabolism, it is again perhaps surprising that the adrenal ZF is par- ticularly susceptible to mutations in one of the antioxidant defense systems. Increased R OS levels are known to be deleterious and steroidogenic cells are exposed to high levels because of the use of molecular oxygen in steroido- genesis and the production of free radicals during CYP reactions (Hornsby, 1989; Hornsby & Crivello, 1983). Again, because of the relatively high levels of steroidogenesis in the ZF compared to the ZG and other steroido- genic tissues, and the critical importance of glucocorticoids for health, impairment of these cells may be noticed before effects on other tissues. Interestingly, the C57Bl6/J strain harboring the Nnt mutation develops glu- cose intolerance, and it may be necessary to monitor FGD patients with NNT mutations for extra-adrenal complications.
These causes of FGD still only explain just over 60% of all cases, and the involvement of cell cycle and antioxidant defense genes indicates that the adrenal cortex is particularly susceptible to the impairment of seemingly common systems. It is anticipated that the discovery of the causes of the remaining cases of FGD will give further insight into the control of adrenal steroidogenesis and ZF maintenance.
9.3. Triple-A syndrome
Triple-A syndrome, also known as Allgrove syndrome, is an autosomal recessive disease characterized by adrenal insufficiency together with
achalasia of the cardia and alacrima (Huebner et al., 2000). The gene respon- sible, AAAS, encodes a nuclear pore protein named ALADIN (Handschug et al., 2001; Tullio-Pelet et al., 2000) and mutations in this gene cause ALADIN to sequester in the cytoplasm (Cronshaw & Matunis, 2003) and defective nuclear transport of DNA repair enzymes and antioxidant defense proteins (Hirano, Furiya, Asai, Yasui, & Ueno, 2006; Storr et al., 2009). Fibroblasts from these patients are susceptible to oxidative stress, and the pathogenesis of AAAS for the adrenal gland may be similar to that observed for Nnt, adding further support to the importance of controlling antioxidant levels for adrenal viability.
9.4. Adrenal cancer
Adrenocortical tumors are common, with the majority, referred to as incidentalomas, being asymptomatic and revealed during investigations for other disorders or at autopsy. These benign, nonmetastatic, adenomas usually remain undiagnosed unless they are very large or hypersecrete hor- mones such as cortisol-producing adenomas (CPAs) in Cushing’s syndrome and aldosterone-producing adenomas (APAs) in Conn’s syndrome. Adreno- cortical carcinomas, by contrast, are rare (between 1 and 12 per million adults; Grumbach et al., 2003) and malignant with a poor prognosis and a median survival time of less than 12 months from detection (Sidhu, Sywak, Robinson, & Delbridge, 2004), but they can also be secreting or nonsecreting. It is as yet unclear whether adenomas can progress to become carcinomas or whether adenomas and carcinomas are diseases with different etiologies.
Steroid-secreting adrenocortical carcinomas have increased expression levels of steroidogenic genes, CYP11B2 in APAs and CYP11B1 in CPAs, as well as increased SF-1 levels (Bassett et al., 2005; Boulkroun et al., 2013). Analysis of adrenalectomies from patients with APAs indicates that both the tumor and peritumoral tissues express more CYP11B2, indicating a remo- deling of the ZG, although transcriptomic analyses of the peritumoral tissue do not support a mechanism suggesting that the remodeling is an initial step towards APA formation. APAs often have the histological appearance and transcriptional profile of ZF cells rather than ZG cells, but whether they are ZG cells that have acquired ZF characteristics or ZF cells that have acti- vated markers of ZG expression, such as CYP11B2 and Dab2, is currently controversial but interesting for the etiology of tumor formation (Azizan et al., 2012; Boulkroun et al., 2013). Somatic and germline mutations in
KCNJ5, a gene encoding a potassium channel; GIRK4 (Boulkroun et al., 2012, 2013; Choi et al., 2011); and somatic mutations in two ATPases, ATP1A1 encoding the x1-subunit of the Na+/K+ ATPase and ATP2B3, encoding the plasma membrane calcium-transporting ATPase 3, have been identified in approximately 7% of APAs (Beuschlein et al., 2013). These pro- teins are involved in maintaining cell membrane potential, and mutations lead to excess aldosterone production as a result of membrane depolarization and calcium influx, which activates CYP11B2 expression and aldosterone synthesis.
Other inherited forms of adrenocortical carcinoma include Li-Fraumeni syndrome, MEN-1, and Beckwith-Wiedemann syndrome (BWS). Li-Fraumeni syndrome is an autosomal dominant syndrome of cancers including osteosarcomas, soft tissue sarcomas, breast cancer, and adrenocor- tical carcinoma (Li & Fraumeni, 1969), as a consequence of germline muta- tions in TP53, which encodes the p53 tumor suppressor protein (Varley et al., 1999). The frequency of childhood adrenocortical carcinoma is highest in southern Brazil and associated with a germline TP53 mutation that does not appear to cause other cancers in the Li-Fraumeni syndrome (Ribeiro et al., 2001). Interestingly, these tumors display very high chromo- somal damage including frequent amplification of the 9q33-q34 locus that contains the SF-1 gene (Figueiredo et al., 1999), and overexpression of SF-1 in mice causes hyperplasia and adrenocortical cancer (Doghman et al., 2007), although the tumors have a gonadal phenotype (see later). TP53 mutations have also been observed in sporadic adrenocortical carcinomas but not ade- nomas, suggesting that TP53 mutation could be an important event in malignant metastatic adrenal cancer.
BWS is a pediatric overgrowth syndrome with features comprising macrosomia, abdominal wall defects, macroglossia, gigantism, and child- hood tumors. In 5% of cases, these include adrenocortical carcinoma (Elliott, Bayly, Cole, Temple, & Maher, 1994). BWS is principally caused by misregulation of an imprinted region on the short arm of chromosome 11 at 11p15 that includes the maternally expressed IGF2 and paternally expressed CDKN1C and H19 genes. Inheritance of two copies of the pater- nal 11p15 allele (paternal uniparental isodisomy), leading to overexpression of IGF2 and loss of expression of p57Kip2, is observed in 10-20% of BWS, and mutations of CDKN1C account for 5-10% of cases (Jacob, Robinson, & Lefebvre, 2013). Increased IGF2 levels are commonly observed in adrenocortical carcinoma, but not adenomas, and there is a pos- itive correlation between levels of expression, malignancy, and recurrence
rates (Boulle, Logie, Gicquel, Perin, & Le Bouc, 1998; Gicquel et al., 2001). TP53 mutations can also lead to the upregulation of IGF2 as seen in Li- Fraumeni syndrome (Ribeiro & Figueiredo, 2004). Several lines of evidence have indicated the importance of IGFs in adrenal growth and differentiation; IGF2 levels are high during fetal adrenal development and IGF2 transgenic mice have large adrenal glands with hyperplastic ZFs, and other components of the IGF system, namely, the receptor IGFR1 and the binding protein IGFBP2, but not IGF1, are also upregulated in ACC (Boulle et al., 1998; Weber, Auernhammer, Kiess, & Engelhardt, 1997). Tp53 mutant mice (Else et al., 2009) or those in which IGF2 is overexpressed (Drelon et al., 2012; Heaton et al., 2012) do not develop adrenal tumors, however, indi- cating these events in themselves are not sufficient for neoplasia.
9.4.1 Wnt/B-catenin
As described earlier, canonical Wnt signaling plays an important role in adre- nal development, and it also appears to be involved in adrenal adenoma and carcinoma. Increased cytoplasmic and/or nuclear ß-catenin is a frequent observation in both adenomas and carcinomas (Bonnet et al., 2011; Tissier et al., 2005), and mutations in ß-catenin are often observed in adre- nocortical tumors (Tadjine, Lampron, Ouadi, & Bourdeau, 2008). These mutations cluster in exon 3 of CTNNB1 that encodes amino acids that are required for targeting the protein for destruction, in particular serines and threonines that are phosphorylated by the destruction complex or amino acids adjacent to these phosphorylated residues, with mutation of serine 45 particularly common in adrenocortical tumors. These serve as activating mutations of ß-catenin as it is no longer degraded and can accumulate in the cytoplasm and nucleus and activate TCF/LEF-mediated transcription. Biallelic inactivating mutations in APC have also been observed in patients with adrenal tumors and familial adenomatous polyposis, and together, these data indicate that Wnt/ß-catenin pathway activation is an important event in adrenocortical tumor etiology. Histological studies indicate that there is greater ß-catenin accumulation in adrenocortical carcinomas than adenomas and nuclear localization of ß-catenin is associated with a more aggressive form of cancer and a poorer prognosis (Gaujoux et al., 2011). Pathway mutations are observed more frequently in larger, nonsecreting adenomas, suggesting that they produce less differentiated and more aggressive forms of tumor (Bonnet et al., 2011). Adrenal-specific expression of an active form of B-catenin (Acat; Berthon et al., 2010) in mice has recently been shown to cause adrenal hyperplasia, confirming a role for this pathway in tumor
initiation, but only a third of these animals developed malignant tumors, implying that other events were required to trigger this transformation. Although activation of the Wnt/ß-catenin pathway appears to be corre- lated with adrenal tumor formation, ß-catenin-activating mutations and APC-inactivating mutations are only found in a minority of carcinomas (26%; Gaujoux et al., 2010). Mutations in Axin2 have recently been described in a small number of both adrenal adenomas and carcinomas, but alterations in other pathway components such as downregulation of extracellular inhibitors of the pathway or overexpression of Wnts could underlie this phenomenon. One potential factor is IGF2, which is known to activate ß-catenin signaling in vitro by inhibiting GSK3ß phosphoryla- tion (Cross, Alessi, Cohen, Andjelkovich, & Hemmings, 1995) and pro- moting direct phosphorylation of ß-catenin on serine 552 via AKT that inhibits degradation (Fang et al., 2007). When ß-catenin was activated and IGF2 overexpressed together in mouse adrenal glands, hyperplasia was observed earlier and progressed more frequently to adenoma than was seen with active ß-catenin alone and in one case developed into a car- cinoma (Heaton et al., 2012). These data may indicate that activation of the Wnt/B-catenin pathway may be a common event in adrenal cancer and that adenomas do progress to carcinomas if activating mutations in other pathways accumulate.
9.4.2 PKA pathway
Another pathway that is frequently activated in adrenal tumors is the pro- tein kinase A pathway. Inactivating mutations of the PRKAR1A gene that encodes the type 1a regulatory subunit of PKA are found in 10% of adre- nocortical adenomas that tend to be small and pigmented and are classed as primary pigmented nodular adrenocortical disease (PPNAD). PPNAD may be associated with ACTH-independent Cushing’s syndrome and is also a common feature of the autosomal dominant multiple endocrine neoplasia syndrome known as Carney complex. 80% of Carney complex patients with Cushing’s syndrome have mutations in PRKAR1A (Kirschner et al., 2000). LOH of the locus containing PRKAR1A, 17q22-24, is fre- quently observed in adrenal cancer, reported to be observed in 23% of ade- nomas and 53% of carcinomas (Bertherat et al., 2003). Mutations of the gene are less frequent, however, observed in 10% of adenomas and not seen in carcinomas. The adrenal gland-specific knockout of Prkar1a in mice (Sahut-Barnola et al., 2010) recapitulates the main features of PPNAD, ACTH-independent Cushing’s syndrome, and adrenocortical hyperplasia.
Definitively, demonstrating that loss of activity of the R 1x-subunit of PKA results in PPNAD and Carney complex, these studies give insight into the role of the PKA pathway in adrenal development. The hyperplasia observed in these animals appears to arise from the medulla/ZF boundary, with a population of eosinophilic, hypertrophic cells expanding outward from the medulla to the ZG from 5 to 18 months. This cell population resembles the X zone and expresses the X zone marker 20xHSD as well as markers of the ZF. CYP17, which is normally only expressed in the fetal rodent adrenal gland, is also expressed in these cells, and, as a consequence, the mutant adrenal glands make cortisol as well as corticosterone. The mutant adrenal glands express higher levels of inhibin, which has also been observed in human PPNAD nodules, and inhibin is more highly expressed in human fetal than adult adrenal glands (Voutilainen, Eramaa, & Ritvos, 1991). It has been proposed that activins are involved in fetal and X zone apoptosis (see earlier), and one consequence of dysregulated PKA activity may be enhanced expression of inhibin that by competitively inhibiting activin signaling causes the persistence of the fetal/X zone cells leading to adrenal hyperplasia.
Other examples of disorders resulting from PKA pathway dysregulation include McCune-Albright syndrome (MAS), a pediatric disorder not only classified by peripheral precocious puberty, polyostotic fibrous dysplasia, and café-au-lait pigmentation but also associated with other endocrine disorders such as ACTH-independent macronodular hyperplasia (Zacharin, 2007). MAS is caused by a mutation in GNAS1, the gene that encodes the stim- ulatory G protein subunit Gas that activates adenylate cyclase and cAMP- dependent PKA signaling following GPCR stimulation, as is the case in MC2R activation by ACTH. The mutation results in constitutive activation of adenylate cyclase and the PKA pathway. This mutation has been identi- fied in cortisol-secreting adrenal adenomas but not so far in carcinomas. Other mutations in the PKA pathway associated with adrenocortical tumors include PDE11A and PDE8B, genes encoding phosphodiesterases that neg- atively regulate the pathway and have been observed not only in hyperplasia but also in 19% of adenomas and 16% of carcinomas (Libe et al., 2008; Vezzosi et al., 2012). Like AKT, PKA phosphorylates and inactivates GSK3B (Fang et al., 2000) and phosphorylates ß-catenin (Hino, Tanji, Nakayama, & Kikuchi, 2005) to activate ß-catenin-dependent transcription, and ß-catenin accumulation has been reported in PPNAD (Gaujoux et al., 2008). This may again indicate that ß-catenin pathway activation is a requirement for adrenocortical cancer.
9.4.3 Gata4, Gata6, and adrenocortical hyperplasia
An interesting feature shared by some of the mouse models of adrenocortical disease and cancer described earlier is the development of spindle-shaped cell hyperplasia at the periphery of the gland as the animal ages, such as in Mcm4-depleted mice (Hughes et al., 2012), Prkar1a-null mice (Sahut- Barnola et al., 2010), ß-catenin-overexpressing Acat mice (Berthon et al., 2010), SF-1-overexpressing mice (Doghman et al., 2007), and IGF2- overexpressing mice (Drelon et al., 2012). The phenotype of this spindle- shaped cell hyperplasia is very similar to that observed in certain inbred mouse strains that develop adrenocortical tumors following prepubertal gonadectomy. These strains include DBA/2J, CE/J, C3H, Balb/c, IQI/ Jic, and NU/J mice (Bernichtein, Alevizaki, & Huhtaniemi, 2008; Bernichtein, Petretto, et al., 2008; Bielinska et al., 2006; Kim, Kubota, Kiuchi, Doi, & Saegusa, 1997). Shortly after gonadectomy, densely packed nests of small, spindle-shaped, basophilic cells appear just below the adrenal capsule, referred to as A cells (for reviews, see Parviainen et al., 2007; Vuorenoja et al., 2007). These cells are nonsteroidogenic, expressing neither SF-1 nor other steroidogenic enzymes, but as they invade the steroidogenic cortex in a centripetal manner similar to that observed for normal adrenal cell migration, they become interspersed with larger, rounder, eosinophilic ste- roidogenic cells, referred to as B cells (Parviainen et al., 2007). Human and rodent adult steroidogenic cells express the zinc finger transcription factor Gata6, but both A and B cells express Gata4 (Parviainen et al., 2007), which is normally expressed only in the fetal adrenal glands, and other markers of gonadal cells such as Amhr2 (Krachulec et al., 2012). The steroidogenic B cells express CYP17, required for sex steroid as well as cortisol synthesis, gremlin (Krachulec et al., 2012), and the gonadotropin receptor LHCGR, hallmarks of gonadal-like cells (Bernichtein, Alevizaki, & Huhtaniemi, 2008; Bernichtein, Petretto, et al., 2008; Parviainen et al., 2007; Vuorenoja et al., 2007). Neither A nor B cells express MC2R or other adre- nal steroidogenic markers such as CYP11B1 or CYP11B2, indicating that these cells have adopted a gonadal rather than adrenal identity, and the B cells can secrete sex steroids (Bernichtein, Alevizaki, & Huhtaniemi, 2008; Bernichtein, Petretto, et al., 2008). Gonadectomy interrupts the feed- back of the hypothalamic-pituitary-gonadal (HPG) axis by gonadal sex steroids, and it was likely that the absence of these or the resulting increase in pituitary gonadotropins was responsible for the development of the hyperplastic phenotype. Xenografts of CHO cells engineered to express human chorionic gonadotropin in NU/J mice also caused the development
of spindle-cell hyperplasia, suggesting that it is the elevated gonadotropins that drive the hyperplasia (Bielinska et al., 2005).
Several steroidogenic enzyme promoters contain consensus GATA binding sites, including StAR, 17ß HSD, CYP17, and CYP19 as well as SF-1, with which Gata factors can act in synergy to activate transcription of these promoters in vitro (for reviews, see Parviainen et al., 2007; Tremblay & Viger, 2003), indicating that in the adult adrenal gland, Gata6 can regulate steroidogenic gene expression. Several investigations have demonstrated that the switch in expression from Gata6 to Gata4 is causative in the development of hyperplasia. First, mice in which Gata6 is condition- ally deleted from steroidogenic cells, as well as displaying a thin cortex with a thickened capsule, also develop Gata4-positive gonadal-like spindle-cell hyperplasia that is enhanced by gonadectomy, with the accumulation of LHCGR- and CYP17-positive B cells (Pihlajoki et al., 2013). Gata4- haploinsufficient B6D2F1 mice are resistant to gonadectomy-induced adrenal hyperplasia, and Amhr2 cre-mediated Gata4 deletion in these mice produces only small nests of A cells following gonadectomy that do not extend far into the cortex and do not develop into B cells, indicating that Gata4 expression is necessary for both gonadectomy-induced adrenal neo- plasia and its progression and not just a consequence of it (Krachulec et al., 2012). Furthermore, overexpression of Gata4 in mice under the control of the Cyp21 promoter causes spindle-cell hyperplasia in females, which appears in males and is increased in females following gonadectomy, leading to adenoma formation (Chrusciel et al., 2013).
Two other mouse models that develop adrenal tumors following gonad- ectomy are the inhibin a-null mouse (Matzuk, Finegold, Su, Hsueh, & Bradley, 1992) and the Inha/Tag mouse in which SV40 large T antigen is expressed under the control of the inhibin a promoter (Kananen et al., 1996, 1997). These tumors are also Gata4-positive and express gonadal markers and secrete sex steroids, but unlike in the models mentioned earlier, they appear to form at the medulla boundary, although spindle-shaped cells have been observed at the periphery in some cases, and it has been proposed that the tumors arise from these subcapsular cells (Looyenga & Hammer, 2006). The Prkar1a mouse, as well as developing central tumors composed of large SF-1-positive steroidogenic cells, also displays small subcapsular spindle-shaped cells (Sahut-Barnola et al., 2010). Furthermore, this is also seen in the Acat constitutively active ß catenin mouse model (Berthon et al., 2010). Again, two kinds of hyperplastic cells, SF-1-positive steroido- genic cells found in the inner zones of the cortex and in the central medulla
region, which go on to form a carcinoma as the mice age, and basophilic, SF-1-negative and Gata4-positive, spindle-shaped cells at the periphery of the gland, which by 10 months have traversed the cortex to reach the medulla. Interestingly, these cells do not express ß-catenin, suggesting that B-catenin activation has indirect effects on other cell populations that give rise to these cells. In the Mcm4 mouse model, spindle-shaped cells had infil- trated the cortex from the subcapsular region by 3 months of age and by 12 months had spanned the cortex from the capsule to the medulla (Hughes et al., 2012). These were phenotypically equivalent to A cells, expressing Gata4 but no steroidogenic markers or LHCGR. They were, however, both ß-catenin- and Gli1-positive, and interestingly, the capsule was markedly thinned adjacent to the areas of infiltrating hyperplastic cells (Fig. 7.5). Loss of Mcms in mouse models is associated with stem cell defects (Chuang, Wallace, Abratte, Southard, & Schimenti, 2010; Pruitt, Bailey, & Freeland, 2007), and the apparently specific impingement of the MCM4 defect on adrenal function may be a consequence of its effect on the growth and differentiation of these putative capsular stem/progenitor cells.
Taken together, these data may indicate that the central tumors arise from fetal/X zone cells, whereas the subcapsular spindle-shaped cells may result from a misspecification of capsular or subcapsular mesenchymal stem cells that, rather than differentiating to enter the steroidogenic lineage as Gata6-positive cells, instead express Gata4 and retain Gli1 expression. In support of this hypothesis is the observation that Gli1 expression was also elevated in adrenal glands with hyperplastic spindle-shaped cell infiltration in Gata6-null mice (Pihlajoki et al., 2013). Furthermore, overexpression of
Capsule
Capsule
Capsule
A cell
A cell
B cell
IGF2 in mouse adrenal glands also led to the production of subcapsular Gata4-positive spindle-shaped cells that expressed Gli1 and another capsule marker, Pod1 (Drelon et al., 2012). Interestingly, even though the Cyp21 promoter was used to overexpress Gata4 in the adrenal gland (Chrusciel et al., 2013), no CYP21-positive cells and spindle-shaped cells were observed. This may indicate that the neoplastic cells are derived from differ- entiating stem cells that transiently activate CYP21 expression before it is extinguished by the Gata4 overexpression. This view is supported by the formation of subcapsular spindle-cell hyperplasia in the Prkar1a conditional-null, ß-catenin, and IGF2-overexpressing mouse models in which the genetic recombinations are controlled by ark 1b7 cre. This cre is only expressed at high levels in the ZF (Lambert-Langlais et al., 2009; Sahut-Barnola et al., 2010), again suggesting that the subcapsular spindle- shaped cell hyperplasia is likely be a non-cell-autonomous effect of these modifications. Indeed, the spindle-shaped cells do not express ß-catenin in the Acat mouse (Berthon et al., 2010).
Are these observations relevant for human disease? It is interesting to note that adrenal tumorigenesis in humans sometimes occurs following dis- ruption of the HPG axis, for example, in postmenopausal women (Mijnhout, Danner, van de Goot, & van Dam, 2004; Pabon et al., 1996) and men with testicular atrophy (Romberger & Wong, 1989). These patients have marked elevation in gonadotropins, a situation equivalent to that in the gonadectomized animals. Furthermore, subcapsular spindle-cell tumors are reported in some of these cases (Fidler, 1977), and Gata4 and LHCGR expression has been detected as well as downregulation of Gata6 (Barbosa, Giacaglia, Martin, Mendonca, & Lin, 2004; Kiiveri et al., 2005; Saner-Amigh et al., 2006). Interestingly, male patients with congenital adre- nal hyperplasia (CAH) have a high incidence of testicular tumors, called adrenal rests, which have features of adrenal steroidogenic cells, expressing MC2R, CYP21, and CYP11B1 (Claahsen-van der Grinten, Otten, Stikkelbroeck, Sweep, & Hermus, 2009; Claahsen-van der Grinten, Stikkelbroeck, Sweep, Hermus, & Otten, 2006). CAH is predominantly caused by mutations in the cortisol pathway synthetic enzymes CYP21 or CYP11B1, such that the HPA axis negative feedback is impaired resulting in elevated levels of ACTH (Maitra & Shirwalkar, 2003). Perhaps, excess ACTH or gonadotropins can direct the differentiation of stem cells in the gonads or adrenal glands down an adrenal or gonadal pathway, respectively, possibly by upregulating very low levels of MC2R or LHR expression in these cells. If the spindle-shaped cells described earlier do arise from the
adrenal capsular stem/progenitor cells but adopt the phenotype of gonadal stromal cells, it raises the intriguing possibility that the stem/progenitor cells have the ability to convert to a gonadal as well as an adrenal identity. It cer- tainly appears likely that the elevated LH levels following gonadectomy are required for the development of B cells, with perhaps a feedforward mech- anism with Gata4 upregulating LHCGR expression, although this has not been demonstrated. An alternative explanation is that the segregation of the adrenal and gonadal primordia is not a completely efficient process and these inappropriately localized cells can respond to tropic stimuli. ACTH-responsive CYP11B1- and CYP21-positive cells have been observed in mouse testes from 13.5 dpc through to adulthood, perhaps in support of this idea (Val, Jeays-Ward, & Swain, 2006).
10. SUMMARY
Our understanding of the control of adrenal development has increased over the last few years with the increasing use of animal models and lineage-tracing techniques, but there is still much left to be discovered. We have yet to definitively identify adrenocortical stem/progenitor cells and it is likely that these cell populations have important roles in adult homeo- stasis and disease, another important area for study. The constant remodeling of the zones of the adrenal cortex presumably requires the precise control of the growth of these stem/progenitor populations and their differentiation into the appropriate mature steroidogenic cells. We do not know whether these are principally in the capsule or in the subcapsular cortex or whether both populations can play a role. Nor do we know the relative contributions of Shh and Wnt/ß-catenin signaling or how these may be controlled by other signals, including those from the RAAS and HPA axes. The mecha- nisms that control zonation, such as positional cues during centripetal migra- tion, are also undetermined at present. Understanding these processes and the mechanisms controlling them will help to elucidate the causes of adrenal insufficiency and cancer, conditions that are not optimally managed at pre- sent. Ultimately, identification of the stem cells and understanding their ori- gins and how the balance between self-renewal and differentiation is maintained may allow targeted cell replacement approaches to treat adrenal insufficiency disorders that require lifelong hormone replacement therapies. Identifying novel pathways to target for treatment of adrenocortical carci- noma is a particularly important therapeutic goal.
It is less than 20 years since the discovery that deletion of Sf-1 caused adrenal agenesis, a seminal publication that ushered in an era of investigation of the mechanisms controlling adrenocortical development and function. It can be hoped that at the current rate of progress, it will not be too long before these and other questions have been answered.
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