Adrenal Cortex Hormones

C H A P T E R

28 Adrenal Cortex Hormones Nicolas C. Nicolaides1,2, George P. Chrousos1,2 1

Division of Endocrinology, Metabolism and Diabetes, First Department of Pediatrics, National and Kapodistrian University of Athens Medical School, ‘Aghia Sophia’ Children’s Hospital, Athens, Greece; 2Division of Endocrinology and Metabolism, Center of Clinical, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

1. ADRENAL GLANDS: ANATOMY AND HISTOLOGY

contributing substantially to the maintenance of our resting and stress-related homeostasis (Stewart, 2003; Miller, 2005; Nicolaides et al., 2014).

The adrenal glands are endocrine organs, located in the retroperitoneum on the upper poles of the kidneys. They are surrounded by a stroma of connective tissue, termed “capsule,” which contributes to the maintenance of adrenal structure. The adrenal glands consist of two functionally and embryologically distinct parts, the adrenal cortex and the adrenal medulla, which influence each other’s development, growth, and endocrine function (Stewart, 2003; Miller, 2005; Nicolaides et al., 2014). The adrenal cortex originates from the mesoderm and is composed of the outer definitive zone and the inner fetal zone. The fetal zone occupies 80% of the adrenal volume during fetal life and secretes dehydroepiandrosterone (DHEA). Following delivery, this zone regresses rapidly during the first 2 weeks of life and disappears by the third month. Within the next 3 years, the definitive zone and remnants of the fetal zone give rise to the adrenal cortex consisting of three layers: the outer zona glomerulosa, the intermediate zona fasciculata, and the inner zona reticularis. The zona glomerulosa contains aldosterone-secreting cells, which are scattered under the adrenal capsule. The zona fasciculata represents the site of cortisol production and consists of large cells with more lipids, the “clear cells.” The zona reticularis is responsible for the production of adrenal androgens. This zone is narrow and contains the “compact” cells, which are full of lipofuscin granules (Stewart, 2003; Miller, 2005; Nicolaides et al., 2014). The adrenal medulla comprises 10% of the adrenal weight and is functionally related to the activity of the sympathetic nervous system. This anatomic part of the adrenal glands produces epinephrine and norepinephrine,

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00028-6

2. BIOSYNTHESIS OF ADRENAL CORTEX HORMONES The biosynthesis of adrenal cortex hormones is regulated by independent systems. The production and release of cortisol are controlled by adrenocorticotropic hormone (ACTH), whereas aldosterone biosynthesis and secretion are regulated by the renin-angiotensin system and circulating potassium concentrations. All adrenal cortex hormones are derived from cholesterol (Fig. 28.1). About 80% of cholesterol utilized in biosynthesis of these hormones originates from plasma low-density lipoprotein, while a small amount of free cholesterol molecules is produced by enzymatic hydrolysis of stored cholesterol esters in adrenal steroidogenic cells. In addition, an increased uptake of plasma lipoproteins from peripheral circulation occurs upon stimulation of adrenal cortex cells by several signals (Stewart, 2003; Miller, 2005; Nicolaides et al., 2014; Simpson and Waterman, 1995). These mechanisms contribute substantially to the availability of cholesterol for steroidosynthesis by adrenocortical cells. During adrenal steroidogenesis, cholesterol undergoes several structural changes, which are caused by serial enzymatic reactions. The responsible enzymes, termed “steroid hydroxylases,” belong to the cytochrome P450 (CYP) superfamily. The first and rate-limiting step in steroid biosynthesis is the import of cholesterol molecules from several stores to the matrix side of the mitochondrial inner membrane, a process controlled by the

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28. ADRENAL CORTEX HORMONES

Cholesterol

CYP11A1

Progesterone

Pregnenolone

11-Deoxy-corticosterone CYP21

HSD3B1

Corticosterone

CYP11B1

Aldosterone

CYP11B2

CYP17

CYP17

17-OH Pregnenolone

17-OH Progesterone

Cortisol

11-deoxycortisol CYP21

HSD3B1

Cortisone HSD11B1

CYP11B1

CYP17

DHEA

Estrone

Androstenedione HSD3B1

CYP19

HSD17B1

HSD17B2

HSD17B1

Androstenediol HSD3B1

17β -Estradiol

Testosterone

DHT

CYP19 5α -Reductase

FIGURE 28.1

Biosynthesis of adrenal cortex hormones. CYP, cytochromes P450; HSD, hydroxysteroid dehydrogenase.

steroidogenic acute regulatory (StAR) protein. Of note, the production of StAR is regulated by several signals, including ACTH (Simpson and Waterman, 1995; Stocco and Clark, 1996; Arakane et al., 1998). Cholesterol import is also mediated by the peripheral benzodiazepine receptor (Papadopoulos, 1998). The first enzymatic reaction takes place in the mitochondrion and is catalyzed by the cytochrome P450 enzyme CYP11A (P450scc, cholesterol desmolase, side chain cleavage enzyme) leading to the cleavage of six carbon atoms from the side chain of cholesterol, thereby converting the C27 compound to the C21 steroid pregnenolone (Nebert et al., 1991). The latter leaves the mitochondrion to the cytoplasm, where it undergoes further metabolism by other enzymes. To produce mineralocorticoids in the zona glomerulosa, the microsomal 3b-hydroxysteroid dehydrogenase (3b-HSD) converts pregnenolone to progesterone (Cherradi et al., 1997). Progesterone then undergoes 21hydroxylation in the cytoplasm by CYP21 (P450c21, 21-hydroxylase) to produce deoxycorticosterone (DOC). DOC is 11b-hydroxylated to produce corticosterone, which undergoes serially 18-hydroxylation and 18-oxidation to finally form aldosterone. The two hydroxylations and the final oxidation are catalyzed by

the P450 enzyme CYP11B2 (P450aldo, aldosterone synthase) (Fig. 28.1) (White et al., 1994). For the adrenal cortex zona fasciculata to produce cortisol, the enzyme CYP17 (P450c17, 17a-hydroxylase/17,20-lyase) catalyzes the conversion of pregnenolone to 17a-hydroxypregnenolone within the endoplasmic reticulum (Yanase et al., 1991). In the zona fasciculata, 3b-HSD converts 17a-hydroxypregnenolone to 17a-hydroxyprogesterone, which then undergoes 21-hydroxylation by CYP21 to produce 11deoxycortisol. The latter is converted to cortisol, a reaction catalyzed by CYP11B1 (P450c11, 11b-hydroxylase) in the mitochondria (Fig. 28.1). The biosynthesis of adrenal androgens takes place in the zona reticularis and begins with the 17a-hydroxylation of pregnenolone and progesterone. 17a-hydroxypregnenolone is converted to DHEA by the enzyme CYP17. DHEA is then converted to androstenedione by 3b-HSD. In the gonads, androstenedione is reduced by 17b-hydroxysteroid dehydrogenase to produce testosterone (Penning, 1997). In pubertal ovaries, aromatase (CYP19, P450c19) can convert androstenedione and testosterone to estrone and estradiol, respectively (Simpson et al., 1994). Testosterone may be further

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4. GLUCOCORTICOIDS

metabolized to dihydrotestosterone by steroid 5a-reductase in androgen target tissues (Fig. 28.1) (Wilson et al., 1993). Adrenal cortex hormones signal through their cognate receptors; glucocorticoids mediate their actions through the glucocorticoid receptor (GR), mineralocorticoids signal via the mineralocorticoid receptor (MR), whereas adrenal androgens exert their effects through the androgen receptor (AR). All these receptors belong to the steroid receptor subfamily of the nuclear receptor family of transcription factors (Laudet et al., 1992; Thornton, 2001; Baker, 2003).

3. EVOLUTION OF ADRENAL CORTEX HORMONE RECEPTORS In the last 3 decades, scientific interest has focused on the origins of steroid receptors. The fundamental roles of steroid hormone signaling in development, reproduction, intermediary and water metabolism, and maintenance of resting and stress homeostasis provided an early stimulus in cloning and sequencing these proteins from a plethora of organisms, allowing a comprehensive phylogenetic analysis (Laudet et al., 1992). Evolutionary analysis of steroid receptors demonstrated that these proteins form a distinct branch on the phylogenetic tree of nuclear receptors (Laudet et al., 1992). The evolution of steroid receptors consists of two crucial molecular events: gene duplication and spontaneous mutagenesis. Two serial gene duplications of an ancestral steroid receptor took place before the divergence of lamprey and jawed vertebrates (Thornton, 2001). The first gene duplication generated an estrogen receptor and a 3-ketosteroid receptor, whereas the second duplication of the latter gene gave rise to a corticoid receptor (CR) and a receptor for 3-ketogonadal steroids (androgens and/or progestins). Thus, due to two serial gene duplications of an ancestral steroid receptor, three novel receptors emerged: an estrogen receptor, a CR, and a receptor for androgens (AR), progestins (PR), or both (Thornton, 2001). The three receptors duplicated again and yielded six steroid receptors: Estrogen receptor (ER) a and ERb from the ancestral ER, GR, and MR from the CR, and the PR and AR from the 3ketogonadal steroid receptor. The aforementioned gene duplications were followed by random and naturally selected gene mutations providing the steroid specificity in the steroid hormone receptors. In addition to adrenal steroid receptor evolution, steroidogenic and steroid-inactivating enzymes arose at about the same time in primitive vertebrates (Baker, 2003). Indeed, these enzymes are present in fish, which is consistent with the presence of steroid receptors in these organisms (Baker, 2003; Escriva et al., 2000). On

the other hand, the genes encoding these dehydrogenases or cytochrome P450s were not found in the genomes of Caenorhabditis elegans, Drosophila melanogaster, and Ciona, as occurred with the genes of steroid hormone receptors (Baker, 2003; Dehal et al., 2002). Consequently, these enzymes evolved at about the same time as steroid receptors, giving rise to the specific ligands for each steroid receptor.

4. GLUCOCORTICOIDS Glucocorticoids are released into the systemic circulation under the control of the hypothalamic-epituitarye adrenal (HPA) axis in a circadian, ultradian, and stressrelated fashion (Nicolaides et al., 2017). These steroid hormones regulate a plethora of physiologic functions and contribute substantially to the maintenance of resting and stress-related homeostasis. At the cellular level, glucocorticoids play a fundamental role in proliferation, differentiation, and programmed cell death (apoptosis), while accumulating evidence suggests that these hormones influence the methylation status of many cytosine-guanine dinucleotides (CpG), which are located in the regulatory regions of a large number of genes, ultimately leading to epigenetic alterations (Zannas and Chrousos, 2017).

4.1 Regulation of Glucocorticoid Secretion Glucocorticoids represent the end-products of the HPA axis, which responds to many regulatory signals including important negative feedback loops. The HPA axis is composed of three anatomic domains: the paraventricular nuclei (PVN) of the hypothalamus, the pituitary gland, and the adrenal cortex. Several internal or external stimuli, the stressors, trigger the synthesis and secretion of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) from the PVN of the hypothalamus (Chrousos, 1995; Chrousos et al., 1985). CRH is the principal hypothalamic factor that stimulates ACTH secretion, whereas AVP has less ACTH secretagogue activity but synergizes with CRH to potentiate its effects. In addition to CRH and ACTH, several other molecules, including catecholamines, vasoactive intestinal polypeptide, atrial natriuretic peptide (ANP), angiotensin II, pituitary adenylate cyclase-activating polypeptide, and cholecystokinin may also contribute to pituitary ACTH release (Stewart, 2003; Miller, 2005; Simpson and Waterman, 1995). CRH circulates through the hypophysial portal system and reaches the corticotroph cells in the anterior pituitary, where it binds to its cognate G proteinecoupled receptor, the CRHR1 (CRH receptor type 1) (Fig. 28.2).

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28. ADRENAL CORTEX HORMONES Ca

Ca

Ca Ca

CRH

αs

GTP

γ β

αs

GTP

AC

P

cAMP

PKA

Ca

Ca

Ca

Ca Ca

Ca

Rap-1

Ca

CAMKII

Ca

P

CREB

c-Fos

B-Raf MEK1/2 P

ERK1/2

CREB c-Fos

P

Nur77 P

P

POMC

Nur77

CREB c-Fos

NurRE

AP-1

FIGURE 28.2 CRH signaling pathway. AC, adenylyl cyclase; Ca, calcium; cAMP, cyclic AMP; CREB, cAMP response element-binding protein; CRH, corticotropin-releasing hormone; PKA, protein kinase A; POMC, proopiomelanocortin.

Upon binding, the complex CRH-CRHR1 activates the protein kinase A (PKA) through cyclic AMP (cAMP) leading to the activation of two signaling pathways: the mitogen-activated protein kinase (MAPK) pathway and the cAMP response element-binding protein (CREB) pathway (Refojo and Holsboer, 2009). The PKA-induced activation of the MAPK pathway occurs through calcium-dependent and -independent mechanisms, and involves Rap-1, B-Raf, MEK, and ERK1/2. The latter can phosphorylate the orphan transcription factor Nur77, thereby increasing the transcription rate of the POMC (proopiomelanocortin) gene. Upon PKAdependent phosphorylation of the CREB, the latter induces the expression of the c-fos gene through a calcium-dependent mechanism. Both CREB and c-fos bind to regulatory regions of the POMC gene and induce its expression (Fig. 28.2) (Refojo and Holsboer, 2009). POMC is a large precursor molecule of 214 amino acids that proteolytically produces ACTH, b- or g-lipotropins, b-endorphin, a-melanocyte-stimulating hormone (aMSH), amino-terminal peptide, joining peptide, and corticotropin-like intermediate peptide. Importantly, aMSH increases the production of melanin by the melanocytes, leading to skin and mucosal hyperpigmentation

(Stewart, 2003; Miller, 2005; Simpson and Waterman, 1995). Following cleavage of POMC, ACTH reaches the adrenal cortex and binds to high-affinity plasma transmembrane G proteinecoupled receptors and activates adenylyl cyclase (AC), which increases cAMP concentrations (Fig. 28.3). The latter activates PKA, which phosphorylates cholesteryl ester hydrolase (CEH), leading to increased formation of free cholesterol (Nicolaides et al., 2014). In addition, ACTH contributes substantially to a continuous supply of cholesterol to the mitochondria by increasing the uptake of cholesterol from plasma lipoproteins and influencing the remaining steps of steroidogenesis (Fig. 28.3) (Nicolaides et al., 2014). Accumulating evidence suggests that the adrenal cortex may also release glucocorticoids in an ACTHindependent fashion (Bornstein et al., 2008). Thus, an ever-increasing number of molecules, including neurotransmitters, neuropeptides, cytokines, adipokines, growth factors, opioids, and bacterial molecules, interact with specific membrane receptors expressed in adrenocortical cells to induce the release of glucocorticoids independently of ACTH. In addition, paracrine communication between adrenocortical cells and chromaffin

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4. GLUCOCORTICOIDS

ACTH

αs

γ

αs

β

cAMP

PKA

Cholesterol Esters

CEH Cholesterol

Pregnenolone

Lipid Droplet

Cholesterol

17-OH Pregnenolone Pregnenolone scc

3β-HSD

Pregnenolone

17-OH Progesterone

21-Hydroxylase

11-Deoxycortisol 11-Deoxycortisol

11-Deoxycortisol Cortisol Endoplasmic Reticulum

Cortisol

Mitochondrion

FIGURE 28.3 ACTH signal transduction. AC, adenylyl cyclase; ACTH, adrenocorticotropic hormone; cAMP, cyclic AMP; CEH, cholesteryl ester hydrolase; PKA, protein kinase A. Modified from Nicolaides, N.C., Charmandari, E., Chrousos, G.P., 2014. Adrenal steroid hormone secretion: physiologic and endocrine aspects. In: Reference Module in Biomedical Sciences. Elsevier.

cells, nerve endings, and immune cells influences glucocorticoid secretion (Bornstein et al., 2008).

4.2 Glucocorticoid Receptor Glucocorticoids exert their pleiotropic functions through an intracellular, ubiquitously expressed receptor, the GR encoded by the NR3C1 gene (Nicolaides et al., 2010). This gene is located at chromosome 5 and consists of 10 exons, of which exons 2e9a or -9b express the functional protein. Exon 1 contains important regulatory regions that play an important role in gene expression (Nicolaides et al., 2010). In human cells, the alternative use of exons 9a or 9b generates two main protein isoforms, the hGRa or the hGRb, respectively. hGRa is expressed in almost every tissue apart from the SCN of the hypothalamus. At the cellular level, the hGRa is primarily localized in the cytoplasm and, to a lesser extent, in the nucleus and plasma membrane. It binds natural and synthetic glucocorticoids with high

affinity, and regulates gene expression directly by binding to the regulatory regions of glucocorticoidresponsive genes or indirectly through physical interactions with other transcription factors (Nicolaides et al., 2017). On the other hand, the hGRb is expressed in certain cell types, including endothelial cells, and it is primarily localized in the nucleus, possibly functioning as dominant negative inhibitor of hGRa-induced transcriptional activity (Bamberger et al., 1995; Yudt et al., 2003; Charmandari et al., 2005) or influencing the transcription of a number of genes independently of hGRa (Kino et al., 2009a, 2009b). The hGRa consists of eight distinct receptor isoforms with variable N-terminal domains, the hGRa-A (classic GRa), hGRa-B, hGRa-C1, hGRa-C2, hGRa-C3, hGRa-D1, hGRa-D2, and hGRa-D3, which are currently under intense investigation in terms of function and intracellular localization (Oakley and Cidlowski, 2013; Lu and Cidlowski, 2005). We speculate that the same phenomenon occurs with the hGRb isoform. The hGRa and the hGRb are identical

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until the amino acid 727. The hGRa has an additional 50 amino acids, whereas the hGRb has 15 additional, but nonhomologous amino acids (Nicolaides et al., 2010). The hGR is a modular protein, which consists of four domains: the amino-terminal domain (NTD) or immunogenic domain, the DNA-binding domain (DBD), the hinge region (H), and the ligand-binding domain (LBD). The NTD contains a transactivation domain, which is termed “activation function (AF)-1,” between amino acids 77 and 262. The AF-1 is ligandindependent and is used as a platform for the functional interaction between the hGRa and coactivators, chromatin modulators, and basal transcription factors (Chrousos, 2004; Zhou and Cidlowski, 2005; Duma et al., 2006). The DBD lies between amino acids 420 and 480 and consists of the highly conserved motif of the two zinc fingers, which tetrahedrally coordinate a zinc atom and are held by four cysteines (Cys). Of note, the first zinc finger contains a short amino acid sequence, termed “the proximal (P) box,” which plays an important role in the recognition of specific DNA sequences. The “distal (D) box” is located in the second zinc finger and provides a weak dimerization interface of the DBD. In addition to the P box and the D box, the DBD of the hGRa contains amino acid sequences involved in receptor dimerization and nuclear translocation (Zhou and Cidlowski, 2005; Duma et al., 2006). The H region links the DBD with the LBD and provides structural flexibility in the receptor homo- or heterodimers, allowing each dimer to interact with several DNA sequences. Finally, the LBD corresponds to amino acids 481e777 and contains a second transactivation domain, the AF-2, which depends on ligand binding. Further to the AF-2, the LBD contains amino acid sequences that play an important role in dimerization, nuclear translocation, and interaction with heat shock proteins (HSPs) and coactivators (Zhou and Cidlowski, 2005; Duma et al., 2006). The LBD of hGRa contains 12 a-helices and four small b-strands that form a three-layer helical sandwich (Nicolaides et al., 2010). Helices 1 and 3 lie on one side, whereas helices 7 and 10 form the opposite side. Helices 4, 5, 8, and 9 form the middle layer and are located in the top half of the receptor. This structural conformation of helices forms a cavity located in the bottom half of the LBD, allowing natural and synthetic glucocorticoids to bind to the receptor (Bledsoe et al., 2002). Helix (H) 12 contributes substantially to the formation of the LBD and the AF-2. Indeed, upon ligand-binding, the hGRa undergoes such conformational changes that change the position of H11 and H12, allowing coactivators to interact with the AF-2 of the receptor via their LXXLL motif (L stands for leucine and X for any amino acid) (Lu and Cidlowski, 2005). Specifically, upon agonistbinding, the H12 holds a position that permits the

interaction between coactivators and the coactivator cavity (Fig. 28.4). On the other hand, upon antagonist binding, the helical structure in the C-terminal portion of H11 is lost, and the H12 moves over the ligandbinding pocket, therefore preventing coactivator binding and permitting corepressor binding (Fig. 28.4) (Kauppi et al., 2003). The H12 is followed by a strand, which forms a conserved b sheet with a b strand between helices 8 and 9. This strand is crucial for stabilizing H12 in the active conformation (Bledsoe et al., 2002). It also plays an important role in ligand-binding specificity and agonist potential (Kauppi et al., 2003; Zhang et al., 1996).

4.3 Glucocorticoid Signaling In the glucocorticoid target cells, the hGRa is primarily cytoplasmic, forming a multiprotein complex that consists of HSPs and immunophilins (Fig. 28.5). Upon ligand binding, the hGRa dissociates from these proteins and translocates into the nucleus, where it forms homo- or heterodimers that recognize specific DNA sequences, termed “glucocorticoid response elements” (GREs), which consist of two 6-nucleotide half sites separated by three nucleotides (GGAACAnnnTGTTCT) (Bamberger et al., 1996). Upon DNA-binding, the hGRa uses the AF-1 and AF-2 as molecular surfaces to interact with coactivators and chromatin-remodeling complexes. Coactivators, such as p300/CBP, p160, and p/CAF, link the DNA-bound hGRa with the transcription initiation complex, therefore facilitating the transduction of the glucocorticoid signal to the RNA-polymerase II. In addition to coactivators, the hGRa also interacts with chromatin-remodeling complexes, including the mating-type switching/sucrose nonfermenting (SWI/ SNF) complex and components of the vitamin D receptor-interacting protein/thyroid hormone receptorassociated protein (DRIP/TRAP) complex (Heery et al., 1997). The ligand-activated hGRa may also Agonist Form

Antagonist Form

Helix12

FIGURE 28.4 Agonist and antagonist form of hGRa LBD. The yellow arrow indicates the position of helix 12.

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4. GLUCOCORTICOIDS

Hypothalamus

CRH, AVP Pituitary

ACTH

Adrenal cortex

Glucocorticoids

Cytoplasm

GR

GR HSPs HSPs GR GR

FKBP MAPK

PI3K

GR cPLA2α

eNOS

GR

Mitochondrion

L-Arginine

GR BMAL1

NO

GR

Nucleus

CLOCK GR

GR GR

GR

GR

GR

GR TF

FIGURE 28.5 Glucocorticoid signaling pathway. ACTH, adrenocorticotropic hormone; AVP, arginine vasopressin; BMAL1, brain and muscle arnt-like protein 1; CLOCK, circadian locomotor output cycle kaput; cPLA2a, cytosolic phospholipase A2 alpha; CRH, corticotropin-releasing hormone; eNOS, endothelial nitric oxide synthetase; FKBP, immunophilins; GR, glucocorticoid receptor; HSP, heat shock proteins; MAPK, mitogen-activated protein kinases; NO, nitric oxide; PI3K, phosphatidylinositol 3-kinase; TF, transcription factor.

suppress gene expression through binding to negative GREs (nGREs). These DNA sequences have the consensus motif sequence CTCCn0-2GGAGA that differs from the positive GRE in terms of nucleotide sequence and the number of nucleotides between the half sites ranging from 0 to 2 (Hudson et al., 2013). Upon binding to nGREs, the hGRa recruits corepressors, such as NCoR1 and SMRT, and histone deacetylases, ultimately repressing many target genes (Surjit et al., 2011). Therefore, the activated hGRa influences the expression of a large number of glucocorticoidresponsive genes in a positive or negative fashion (Galon et al., 2002). In addition to direct DNA binding, the ligand-bound hGRa can influence the transcription rate of several other genes through proteineprotein interactions with important transcription factors, such as the nuclear

factor-kB (NF-kB), the activator protein-1 (AP-1), and the signal transducers and activators of transcription (STATs) (Fig. 28.5) (Barnes and Karin, 1997; Karin and Chang, 2001; Didonato et al., 1996). During these interactions, the activated hGRa may modulate gene expression without binding to DNA. Further to tethering, the hGRa may bind to composite DNA elements, consisting of a GRE and a response element for a transcription factor, such as STATs or AP-1 (Ramamoorthy and Cidlowski, 2016). These hGRa interactions with the aforementioned transcription factors could explain some of the antiinflammatory actions of synthetic glucocorticoids. In addition to NF-kB, AP-1, and STATs, several in vitro and ex vivo studies have shown that the hGRa interacts with the circadian locomotor output cycle kaput (CLOCK), a transcription factor that influences the circadian oscillation of gene expression (Nader

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28. ADRENAL CORTEX HORMONES

et al., 2009; Nader et al., 2010; Kino and Chrousos, 2011a, 2011b; Charmandari et al., 2011). CLOCK forms a heterodimer with its partner brain and muscle arnt-like protein 1 (BMAL1) and acetylates a cluster of lysine residues within the H region of hGRa, thus leading to reduced DNA binding and decreased sensitivity to glucocorticoids (Fig. 28.5). Therefore, the heterodimer CLOCK/BMAL1 functions as a negative regulator of hGRa transcriptional activity (Nader et al., 2009). More recently, a circadian CLOCK component, termed “chrono” (“ChIP-derived repressor of network oscillator”), was identified as a novel hGRa partner, also linking the circadian clock system and the HPA axis (Annayev et al., 2014; Anafi et al., 2014; Goriki et al., 2014; Robinson, 2014). Further to genomic actions, glucocorticoids exert some of their numerous effects without influencing transcription or translation. These actions are referred to as “nongenomic” and require seconds or minutes to occur. They are mediated mostly by membrane-bound GRs, which trigger kinase signaling pathways, including the MAPK or the phosphatidylinositol 3-kinase (PI3K) (Fig. 28.5) (Groeneweg et al., 2012). Representative examples of nongenomic glucocorticoid actions are the following: (1) some immunosuppressive effects through disruption of T-cell receptor complex (Lo¨wenberg et al., 2006); (2) the rapid vasorelaxation in patients with myocardial or brain ischemia (Hafezi-Moghadam et al., 2002); (3) the rapid increase of frequency of excitatory postsynaptic potentials in the hippocampus that result in altered memory (Karst et al., 2005); and (4) the negative feedback loop at the pituitary level through rapid suppression of ACTH release (Hinz and Hirschelmann, 2000). Glucocorticoids also influence mitochondrial gene expression through GRs that have been detected in these organelles (Fig. 28.5) (Psarra and Sekeris, 2011). The mitochondrial gene expression is influenced directly by mitochondrial GR-GRE interactions and, indirectly, by nuclear GR-GREs interactions, leading to the expression of mitochondrial RNA-processing enzymes, nuclear respiratory factors, or mitochondrial transcription factors (reviewed in Nicolaides and Charmandari, 2017a, 2017b).

5. MINERALOCORTICOIDS Aldosterone plays an important role in the maintenance of fluid volume and sodium and potassium balance, and it regulates blood pressure (Young, 1988). Aldosterone synthesis and release are regulated by the renin-angiotensin system and circulating potassium concentrations. Although ACTH exerts some effect in the zona glomerulosa, angiotensin II has been considered

the most important regulator of aldosterone secretion (Nicolaides et al., 2014).

5.1 Regulation of Mineralocorticoid Secretion Angiotensin II is produced by two serial enzymatic reactions. The first substrate is angiotensinogen, which is synthesized in the liver and secreted into the systemic circulation. In serum, angiotensinogen is cleaved by renin, a protease released by the juxtaglomerular apparatus of the kidney, producing angiotensin I. This reaction is the rate-limiting step for the biosynthesis of angiotensin II (Nicolaides et al., 2014). Angiotensin I is then converted to angiotensin II by angiotensinconverting enzyme. Angiotensin II activates their transmembrane G proteinecoupled receptors (ANGIIR) and inhibits the expression and/or the function of potassium channels and sodium-potassium ATPases, leading to membrane depolarization and subsequent activation of calcium signaling (Fig. 28.6). This signal transduction pathway induces the expression of CYP11B2 gene in the zona glomerulosa and triggers aldosterone synthesis (Beuschlein, 2013). Aldosterone release increases linearly as potassium concentrations rise above 3.5 meq/L (Young et al., 1984). Reduction in sodium concentrations also influences aldosterone secretion, an indirect effect mediated by increased levels of angiotensin II secondary to increased renin concentrations. However, potassium concentrations exert direct effects on aldosterone release through intraadrenal production of angiotensin II. Indeed, zona glomerulosa cells secrete increased concentrations of renin and angiotensin II upon a rise in extracellular potassium concentrations (Kifor et al., 1991; Shier et al., 1989). In addition to angiotensin II and potassium concentrations, ACTH may increase aldosterone secretion through a small increase in intracellular calcium. However, this effect is limited because deoxycorticosterone, which has relatively potent mineralocorticoid activity, is overproduced and because ACTH induces 17a-hydroxylase activity in the zona glomerulosa that converts this compound to cortisol (Nicolaides et al., 2014). On the other hand, ANP has been considered the only known natural inhibitor of aldosterone production (Greenwald et al., 1988).

5.2 Mineralocorticoid Receptor The human mineralocorticoid receptor (hMR) is encoded by the NR3C2 gene, which is located on chromosome 4 in the q31.1 region (Zennaro et al., 1995). The NR3C2 gene consists of 10 exons, of which exons 1a and 1b are untranslated regions, whereas exons 2e9

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5. MINERALOCORTICOIDS

KCNJ5

ANGIIR

Na+,K+ ATPase

Na+,Ca2+ Exchanger

Ca2+ Channel

↑ Sodium Depolarization Calcium Signaling

CYP11B2

Potassium Sodium

Aldosterone Synthesis

Calcium

FIGURE 28.6 Regulation of mineralocorticoid biosynthesis. ANGII, angiotensin II; ANGIIR, angiotensin II receptor; Ca, calcium; K, potassium; KCNJ5, potassium voltage-gated channel subfamily J member 5; Na, sodium. Modified from Nicolaides, N.C., Charmandari, E., Chrousos, G.P., 2014. Adrenal steroid hormone secretion: physiologic and endocrine aspects. In: Reference Module in Biomedical Sciences. Elsevier.

are expressed into a 984-amino acid protein. As it occurs with the hGR, the NR3C2 gene generates several mRNAs translated into functional protein variants (Pascual-Le Tallec and Lombes, 2005). The hMR has four distinct functional domains: (1) the amino-terminal domain (NTD); (2) the central DBD; (3) the hinge (H) region; and (4) the LBD. The hMR NTD is encoded by exon 2 and is the longest domain of the receptor (602 amino acids). This domain has two distinct activation function 1 (AF1) domains, the AF1a (amino acids 1e167) and AF1b (amino acids 445e602), which are responsible for ligand-independent transactivation or transrepression of several MR target genes (PascualLe Tallec and Lombes, 2005). The hMR DBD is encoded by exons 3 and 4, and it consists of 66 amino acids. This region is 94% identical to the hGR DBD and contains the zinc finger motif. The P box is located on the first zinc finger at amino acid positions 621e625, and it is responsible for the binding of the receptor onto the minor groove of the DNA. The D box lies between amino acids 640 and 645 and facilitates receptor dimerization (Viengchareun et al., 2007). The hMR LBD is encoded by exons 5e9 and spans 251 amino acids. It consists of 11 a helices and four antiparallel b strands (Fig. 28.7). Structural biology studies have revealed the crucial role of specific amino acids of the receptor in the interaction of the latter with natural or synthetic ligands. Gln776 of helix H3 and

Arg817 of helix 5 interact with the 3-ketone group of aldosterone. Asn770 of helix H3 contributes substantially to the stabilization of aldosterone 18-hydroxyl group. A specific residue at amino acid position 848 in helix H7 provides ligand specificity between MR and GR (Fig. 28.7) (Li et al., 2005). Moreover, amino acids

FIGURE 28.7 Crystal structure of MR LBD. The arrows indicate the critical amino acid residues in MR LBD.

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820e844 are important for aldosterone binding and ligand binding selectivity (Rogerson et al., 2007). The hMR LBD contains a ligand-dependent AF-2, which consists of the helices H3, H4, H5, and H12. Upon mineralocorticoid binding, the hMR undergoes conformational changes that bring the H12 over the ligand pocket, which, in combination with the bending of helices H3, H5, and H11, forms a hydrophobic cleft on the surface of the LBD (Viengchareun et al., 2007). This structural rearrangement permits the interaction between the hMR and transcriptional coactivators.

5.3 Mineralocorticoid Signaling In the absence of ligand, the hMR is primarily localized in the cytoplasm and like the GR interacts with HSPs and immunophilins (Fig. 28.8) (Lombes et al., 1994). Upon ligand-induced activation, the receptor dissociates from the multiprotein complex and translocates to the nuclear compartment, where it forms homo- or heterodimers and binds to specific hormone response elements, which are located up to 10 kb upstream or downstream from transcriptional start sites in mineralocorticoid-responsive genes. Upon DNA binding, the hMR interacts with coactivators or corepressors to induce or inhibit gene expression (Fig. 28.8). In addition to genomic effects, aldosterone elicits actions in a short time frame. These nongenomic mineralocorticoid actions occur not only in classical MR epithelial target tissues, including kidney and colon,

but also in nonepithelial tissues, such as the heart, adipose tissue, and blood vessels (Ruhs et al., 2017). Several studies have shown that MR is also localized in the plasma membrane, suggesting a pivotal role in aldosterone rapid effects (Callera et al., 2011; Coutinho et al., 2014; Ashton et al., 2015). Since MR does not have a palmitoylation motif that anchors most of the steroid receptors at plasma membrane through palmitic acid, it seems that the membrane association of MR is mediated by scaffolding proteins, which are associated to or inserted in the cell membrane (Ruhs et al., 2017; Grossmann et al., 2010). Indeed, striatin and caveolin-1 are undoubtedly involved in nongenomic MR signaling (Fig. 28.8) (reviewed in Ruhs et al., 2017). Further to direct nongenomic mineralocorticoid signaling, accumulating evidence suggests this rapid signaling pathway crosstalks with receptor tyrosine kinases, including epidermal growth factor receptor, platelet-derived growth factor receptor, and insulin-like growth factor 1 receptor.

6. ADRENAL ANDROGENS In adults, the adrenal cortex produces and releases DHEA and androstenedione in high quantities. Although these steroid molecules are referred to as “adrenal androgens,” they are actually 19-carbon precursors of androgens that have very low affinity for the AR (Nicolaides et al., 2014; Miller, 2017). Small quantities of androstenedione may be used as substrate by 17bHSD5 to

Mineralocorticoids CAV-1 MR HSPs HSPs MR

FKBP

Nongenomic MR signaling

MR

MR

MR

MR

Cytoplasm

MR

MR

MR

Nucleus

FIGURE 28.8 receptor.

Mineralocorticoid signal transduction. CAV-1, caveolin-1; FKBP, immunophilins; HSP, heat shock proteins; MR, glucocorticoid

7. CLINICAL IMPLICATIONS

produce testosterone (Miller, 2017). Of note, recent evidence suggests that the main androgenic steroid released by the adrenal cortex is 11-ketotestosterone (11Keto-T) (Storbeck et al., 2013). Androstenedione and testosterone are converted by P450c11b to 11OHandrostenedione (11OH-D4) and 11OH-testosterone (11OH-T), respectively. Both these 11OH-steroids may undergo oxidation by 11b-hydroxysteroid dehydrogenase type 2 to yield 11-ketoandrostenedione (11KetoD4) and 11-ketotestosterone (11Keto-T), respectively. 11Keto-D4 and 11Keto-T may then be converted to 5aandrostenedione and 5a-dihydrotestosterone, respectively, by the 5a-reductase type 2 (SRD5A2 gene) (Miller, 2017). ACTH has been considered the primary regulatory hormone for the adrenal androgens. The role of adrenal androgens is still under investigation.

7. CLINICAL IMPLICATIONS 7.1 Adrenal Insufficiency Adrenal insufficiency is a potentially life-threatening disorder, which is characterized by defective production or action of glucocorticoids, mineralocorticoids, and/or adrenal androgens (Charmandari et al., 2014). It may be caused by diseases of the adrenal cortex (primary), the pituitary gland (secondary), or the hypothalamus (tertiary). The cardinal clinical features of adrenal insufficiency, as described by Thomas Addison in 1855, include weakness, fatigue, abdominal pain, anorexia, weight loss, hypotension, and salt craving. Patients with primary adrenocortical insufficiency may also present with skin hyperpigmentation (Charmandari et al., 2014; Nicolaides et al., 2017). In adults, the most common cause of primary adrenal insufficiency is autoimmune adrenalitis. In children, primary adrenal insufficiency is often caused by congenital adrenal hyperplasia. Secondary adrenal insufficiency has been linked with diseases that affect the anterior pituitary and interfere with ACTH release. Tertiary adrenal insufficiency is caused by any disease process affecting the hypothalamus and interfering with CRH secretion (Charmandari et al., 2014; Nicolaides et al., 2017; Nicolaides and Charmandari, 2017a, 2017b). Of note, the ever-wider application of next-generation sequencing will undoubtedly enable a deeper and better understanding of novel genetic defects causing adrenal insufficiency.

7.2 Congenital Adrenal Hyperplasia Congenital adrenal hyperplasia (CAH) is a large group of inherited disorders characterized by defects in genes encoding enzymes involved in adrenal

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steroidogenesis (Pretorius et al., 2017). The most common cause of CAH is 21a-hydroxylase deficiency, which accounts for over 90% of all CAH cases. The decrease or complete lack of P450c21 activity leads to a decrease in cortisol biosynthesis and a consequent compensatory increase in ACTH release. The resultant accumulation of cortisol precursors, such as 17-hydroxyprogesterone, drives steroidogenesis toward androgen-producing biochemical pathways leading to in utero virilization in females, rapid somatic growth in both sexes, premature pubarche, precocious puberty, and subfertility (Pretorius et al., 2017; Cabrera et al., 2016). Another less common form of CAH is 11b-hydroxylase deficiency due to CYP11B1 genetic defects. In this form, large quantities of deoxycorticosterone are synthesized. Since this steroid has mineralocorticoid activity, patients with 11b-hydroxylase deficiency may present with hypertension with or without hypokalemic alkalosis. Several other forms of CAH have been attributed to genetic defects that impair adrenal steroidogenesis, including 3b-hydroxysteroid dehydrogenase type 2 deficiency, 17a-hydroxylase deficiency, P450 oxidoreductase deficiency, and others (Nicolaides and Charmandari, 2017a, 2017b).

7.3 Primary Generalized Glucocorticoid Resistance (Chrousos Syndrome) This is a rare endocrinologic condition characterized by partial insensitivity to glucocorticoids in every tissue that expresses the hGR (Chrousos, 2011; Charmandari and Kino, 2010; Charmandari, 2012; Nicolaides and Charmandari 2015, 2017a, 2017b). Tissue resistance to glucocorticoids leads to impaired negative feedback loops at the level of pituitary and the hypothalamus, and this causes compensatory activation of the HPA axis. The increased production of ACTH results in high circulating concentrations of serum cortisol and steroid precursors with mineralocorticoid activity, including corticosterone and 11-deoxycorticosterone, as well as of adrenal androgens. Therefore, patients with Chrousos syndrome may present with depression or anxiety due to high levels of CRH/AVP, chronic fatigue because of increased secretion of cortisol, hypertension with or without hypokalemic alkalosis due to increased concentrations of cortisol precursors, and clinical manifestations of hyperandrogenism, including ambiguous genitalia, virilization, precocious puberty, acne, hirsutism, oligomenorrhea, and decreased fertility. They also have resistance of their HPA axis to dexamethasone suppression, but no clinical evidence of hypo- or hypercortisolism. However, patients with this condition maintain circadian rhythmicity and appropriate responsiveness to stressors, albeit at higher hormone concentrations

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(Chrousos, 2011; Charmandari and Kino, 2010; Charmandari, 2012; Nicolaides and Charmandari, 2015, 2017a, 2017b). Chrousos syndrome has been associated with genetic defects in the NR3C1 gene, such as insertions, deletions, or inactivating point mutations, which impair one or more of the steps of glucocorticoid signaling pathway, thus altering tissue sensitivity to glucocorticoids. To date, 27 different genetic defects have been identified in the NR3C1 gene, the most of which have been identified and extensively investigated by our research group. We and others have demonstrated that all defective GRs impaired several steps of glucocorticoid signal transduction depending on the position of the genetic defect (Chrousos, 2011; Charmandari and Kino, 2010; Charmandari, 2012; Nicolaides and Charmandari, 2015, 2017a, 2017b). However, it is worth mentioning that a number of patients with Chrousos syndrome do not harbor a defective NR3C1 gene, suggesting an important role of newly identified genes in defining tissue sensitivity to glucocorticoids (Nicolaides and Chrousos, 2018).

7.4 Mineralocorticoid Resistance Pseudohypoaldosteronism type 1 (PHA1) is a rare disease characterized by mineralocorticoid resistance. First described by Cheek and Perry in 1958, this condition presents in neonates as a salt-wasting syndrome with vomiting, dehydration, weight loss, and failure to thrive. Despite the extremely increased circulating concentrations of plasma renin and aldosterone, the disease is associated with hyperkalemia and metabolic acidosis (Cheek and Perry, 1958; Zennaro et al., 2012). The first studies aimed to characterize the clinical and molecular mechanisms underlying PAH1 and showed impaired aldosterone binding and signaling in mononuclear cells obtained from patients with PAH1 (Armanini et al., 1985; Kuhnle et al., 1990). Subsequent studies demonstrated the presence of two distinct forms of the disease: a renal form, in which mineralocorticoid resistance is restricted to the kidney, and a generalized form, where mineralocorticoid resistance occurs in the kidney, colon, salivary and sweat glands, and the lungs, presenting as a severe salt-wasting syndrome (Hanukoglu, 1991). The renal form of PAH1 is the most frequent form of the disease and can be found in w1 per 80,000 newborns. Patients with renal PAH1 may present with failure to thrive, dehydration, and vomiting, associated with hyponatremia, hyperkalemia, and increased urinary sodium excretion. Plasma renin and aldosterone concentrations are increased, setting the diagnosis. Symptoms improve in childhood and older children are asymptomatic. The molecular basis of renal PAH1 has been

attributed to inactivating NR3C2 mutations (Zennaro and Fernandes-Rosa, 2017). On the other hand, generalized PHA1, also called autosomal recessive PHA1, is characterized with severe electrolyte disturbances (hyponatremia and hyperkalemia) that may lead to shock, cardiac dysrhythmias, and arrest (Speiser et al., 1986). A number of patients with this form show respiratory tract illnesses and cutaneous lesions (Zennaro and Fernandes-Rosa, 2017). Generalized PAH1 is caused by defects in one of the three genes encoding the subunits of the epithelial sodium channel ENaC (SCNN1A, SCNN1B, and SCNN1G) (Chang et al., 1996; Strautnieks et al., 1996). More than 50 NR3C2 mutations have been reported so far in PAH1 (Arai and Chrousos, 2000; Geller, 2005; Riepe, 2009). These inactivating mutations are located in all exons of the NR3C2 gene, thereby affecting all the functional domains of the receptor (reviewed in Zennaro and Fernandes-Rosa, 2017). The functional characterization of certain NR3C2 mutations located in the DBD or in the LBD has shown that the natural mutant MRs impair several steps of mineralocorticoid signaling depending on the position of the NR3C2 gene mutation (Zennaro and Fernandes-Rosa, 2017).

8. CONCLUSIONS AND FUTURE DIRECTIONS The adrenal cortex consists of three functionally distinct zones, which secrete mineralocorticoids, glucocorticoids, and adrenal androgens. The biosynthesis of these steroid hormones consists of multiple biochemical steps, in which several enzymes play a fundamental role. Each adrenal cortex hormone binds to its cognate receptor, the MR, GR, and AR, all of which belong to the steroid receptor group of transcription factors, influencing gene expression in a positive or negative way, and in a tissue-specific manner. The tremendous progress of molecular and structural biology has enabled the identification of genetic defects in both the enzymes participating in steroidogenesis and the steroid receptors. The application of nextgeneration sequencing technologies will undoubtedly identify novel genetic defects underlying the molecular pathogenesis of steroid- or steroid receptor-associated disorders. Moreover, the ever-increasing use of liquid chromatography followed by tandem MS (LC-MS/ MS) will offer new diagnostic and research opportunities, since this method is vastly more sensitive and accurate than conventional immune-based systems. All this on-going progress resets the adrenal steroidogenesis and steroid signaling as an evolving field in molecular, cellular, and structural endocrinology.

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FURTHER READING

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Further Reading Nicolaides, N.C., Chrousos, G.P., Charmandari, E., 2000e2017. Adrenal insufficiency. In: De Groot, L.J., Chrousos, G., Dungan, K., Feingold, K.R., Grossman, A., Hershman, J.M., Koch, C., Korbonits, M., McLachlan, R., New, M., Purnell, J., Rebar, R., Singer, F., Vinik, A. (Eds.), Endotext [Internet]. MDText.com, Inc., South Dartmouth, MA.