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Division of Endocrinology, Department of Medicine, Research Center, Hôtel-Dieu du Centre Hospitalier de lUniversité de Montréal (CHUM), Montréal, Québec, Canada H2W 1T8
| Abstract |
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I. Introduction
II. Hormonal Regulation of the Normal Adrenal Cortex
III. Primary Adrenal Cushings Syndrome (CS)
IV. Initial in Vitro Evidence of Ectopic Adrenal Membrane Hormone Receptors
V. In Vivo Demonstration of the Functionality of Ectopic or Abnormal Membrane Hormone Receptors
A. Food- and GIP-dependent CS
B. Vasopressin-responsive CS
C. Catecholamine-dependent CS
D. LH-dependent CS
E. LH-dependent adrenal androgen-secreting tumors
F. Serotonin-responsive CS
G. Steroid-responsive CS
H. Other abnormal hormone responses in adrenal CS
VI. Investigation Strategy
A. Initial clinical screening protocol
B. Further characterization of abnormal hormone receptors
C. Systematic clinical screening for ectopic/abnormal hormone receptors
VII. Molecular Mechanisms of Ectopic/Abnormal Hormone Receptors
A. Tissue-specific expression and regulation of membrane hormone receptors
B. Potential mechanisms of ectopic or abnormal hormone receptors
C. Role of ectopic hormone receptors in adrenocortical cell proliferation
VIII. Ectopic/Abnormal Hormone Membrane Receptors in Nonadrenocortical Tumors
IX. An Opportunity for New Pharmacological Therapeutic Strategies
X. Summary and Conclusions
| I. Introduction |
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The mechanisms by which cortisol is produced in adrenal CS, when ACTH is suppressed, were previously unknown and referred to as being "autonomous." Studies by several groups have now shown that some of the cortisol-producing adrenal tumors or hyperplasias may actually be under the control of ectopic (or aberrant, illicit, inappropriate) hormone membrane receptors (8, 9, 10). After a brief overview of the regulation of normal adrenocortical function by its main trophic hormones and of the etiologies of adrenal CS, the present review will focus on in vitro and in vivo findings, identifying abnormalities of expression or function of receptors for various hormones in primary adrenal CS. The mechanisms regulating tissue-specific expression of eutopic membrane receptors in the normal adrenal cortex and the potential molecular alterations leading to the ectopic expression of hormone receptors in adrenocortical tumors and hyperplasias will also be discussed. The identification of abnormal membrane hormone receptors in adrenal CS has now opened the field of new therapeutic strategies to control hypercortisolism by interfering with ligand binding to these receptors and will also be presented.
| II. Hormonal Regulation of the Normal Adrenal Cortex |
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1-adrenoreceptor agonists,
serotonin (5-HT1A) receptor agonists, muscarinic
and nicotinic receptor agonists of acetylcholine, histamine, and
-aminobutyric acid
(GABAA), whereas it
is inhibited by GABAB agonists (14). CRH release
is also stimulated by angiotensin II (Ang-II), neuropeptide Y (NPY),
cholecystokinin (CCK), and gastrin-releasing peptide, or suppressed by
atrial natriuretic peptide (ANP), substance P, somatostatin, and nitric
oxide (NO) (14). Several cytokines, including interleukin-1 (IL-1),
tumor necrosis factor
(TNF-
), and IL-6, stimulate CRH, possibly
through the production of prostaglandins in brain vascular endothelium
(20). ACTH secretion can also be modulated by paracrine/autocrine
interactions, as corticotroph cells have been shown to express CRH,
which can effectively stimulate ACTH release (21).
ACTH binds to its G protein-coupled membrane melanocortin type 2
receptor (22, 23) to elicit short-term (acute) and long-term (chronic)
specific responses, as illustrated in Fig. 1
(24, 25). Activation of the adenylyl
cyclase (AC)/cAMP/cAMP-dependent protein kinase (PKA) pathway leads to
the phosphorylation of proteins that regulate the early and late steps
of steroidogenesis (26, 27). ACTH rapidly (within a few minutes)
promotes the mobilization and transfer of free cholesterol to the inner
mitochondrial membrane (27). Cloning of the steroidogenic acute
regulatory (StAR) protein (28), the subsequent finding of mutations in
the StAR gene responsible for the steroid deficiency disease, lipoid
adrenal congenital hyperplasia (29, 30), as well as the knockout of
this gene in the mouse (31) have identified this ACTH-inducible protein
as a key modulator of cholesterol transport into mitochondria. A second
protein involved in this process is the peripheral-type benzodiazepine
receptor (PBR), which completes the final step of cholesterol delivery
to CYP11A1 (P450scc) for transformation into
pregnenolone (32, 33). ACTH also up-regulates the immediate early genes
c-fos and c-jun via the PKA pathway (25, 34, 35).
A positive feedback loop for the long-term effects of ACTH is
established by the hormone up-regulating its own receptor (36, 37).
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Many ACTH effects are mediated by specific transcription factors (TFs), including orphan nuclear receptors such as nur77 (also called NGFI-B) (39) or steroidogenic factor 1 (SF-1) (40, 41). Indeed, stressful stimuli induce SF-1 and nur77 transcription in corticotrophs and in the adrenal cortex (39, 42). Nur77 and SF-1 both modulate the expression of steroidogenic enzyme genes in the adrenal cortex, nur77 being activated by dephosphorylation and SF-1 by putative PKA-dependent phosphorylation (41, 43, 44).
As an example, SF-1 is involved in the regulation of CYP YP11A (45, 46, 47, 48) and CYP17 (49, 50), where it has been postulated to play a role in constitutive and cAMP-regulated expression. The analysis of the promoter regions of these genes has led to the identification of cAMP-responsive sequences (CRS) and TFs that bind them or synergize cAMP-dependent transcription; general TFs, as cAMP response element (CRE)-binding (CREB) protein and the homeodomain protein Pbx1, both bind CRS and drive cAMP-dependent expression of steroidogenic genes (51, 52, 53, 54, 55). Another ubiquitous TF, Sp1, was shown to regulate basal and cAMP-dependent expression of the CYP11A gene (56). Recent data have suggested that SF-1 is able to mediate cAMP-induced transcription of the CYP17 gene: the proximal CRS (CRS2: 80 to 40) has been identified as a SF-1 binding site (57); moreover, a dominant negative mutation preventing SF-1 binding suppresses cAMP-regulated expression of a reporter gene (58). The coactivator CREB-binding protein (CBP/p300) has been proposed to integrate the effects of TFs such as SF-1, Sp1, CREB, and probably Pbx1 for the regulation of CYP11A and CYP17 genes (59, 60). Moreover, nur77 and nurr1 (nur-related factor 1) positively regulate POMC expression in the pituitary (61, 62). SF-1 up-regulates StAR expression and activity (63). Knockout nur77/ mice demonstrate no remarkable phenotype (64), suggesting that other members of the nur family play redundant roles, perhaps in humans as well. In contrast, SF-1 appears to be essential for the development and survival of steroidogenic organs, as SF-1/ mice lack adrenal glands and gonads and exhibit male-to-female sex reversal of their genitalia (65, 66).
Increasing evidence indicates that adrenocortical steroidogenesis is modulated not solely by ACTH but also by multiple circulating and local peptide hormones, neuropeptides, neurotransmitters, ions, and cytokines (11, 67, 68, 69, 70). Both in vivo and in vitro studies have clearly demonstrated that AVP stimulates aldosterone and cortisol secretion in bovine adrenals (71, 72); in rat cells, AVP stimulates aldosterone but not corticosterone secretion (73, 74). However, it stimulates aldosterone (250%) and cortisol (60260%) secretion from normal human adrenals in vitro (75, 76, 77) via activation of V1-AVP receptors (V1-AVPR) localized mainly in compact cells of the zona reticularis and, to a lesser extent, in the zona glomerulosa (ZG) and fasciculata (68, 74, 78, 79). V2-AVPR were not detected initially in human adrenal cortex tissues (68), but were identified recently by RT-PCR studies (79); their stimulation by DDAVP does not modulate steroidogenesis (79, 80). V3-AVPR (or V1bR) are not detected in the normal human adrenal cortex (79), but are expressed in rat and human chromaffin cells (68, 77, 81), where AVP can stimulate catecholamine release from the adrenal medulla. Thus, AVP could exert significant direct effects on adrenal cortex function, both in endocrine and paracrine modes, but its physiological role has not yet been clearly established. However, in patients with congenital central diabetes insipidus, there is no evidence for clinically significant decreased cortisol secretion (82, 83).
Catecholamines have also been shown to stimulate cortisol and aldosterone secretion in vitro in bovine, pig, and fowl via ß1-adrenoreceptors (11, 84, 85), but this does not appear to occur in human adrenocortical cells (86). Serotonin (5-HT) is another neurotransmitter that may play a role in the control of steroidogenesis (87). 5-HT is able to directly trigger cortisol and aldosterone release, as demonstrated in vitro, in rat, frog, and human adrenal cells (87, 88, 89) but also, indirectly, by stimulating adrenal blood flow (90). The receptor subtype involved in these adrenal effects is still controversial in the rat, but was determined to be 5-HT4 receptor (5-HT4R) in frogs and humans (88, 89). The 5-HT4R is positively coupled to the cAMP and calcium pathways. In vivo, 5-HT4 agonists such as cisapride or zacopride induce an increase in aldosterone but not in cortisol secretion in humans (91, 92). Possible paracrine control of steroidogenesis by 5-HT can be proposed since its presence has been demonstrated in human perivascular mast cells and in chromaffin cells of the frog, rat, and mouse adrenals (93, 94, 95). Central 5-HT is known to enhance ACTH release from the pituitary and to activate the systemic renin-angiotensin system (RAS) to stimulate aldosterone secretion. However, no study has established whether these secretory responses can occur within the adrenal gland in vivo.
VIP and PACAP have been shown to play a paracrine role in the secretory activity of the adrenal cortex in the rat, human, and cow, as they are synthesized by adrenomedullary chromaffin cells (18). VIP stimulates aldosterone release from ZG through the activation of selective VIP receptors (VIPR2/VIPR3), whereas it stimulates cortisol secretion moderately through the nonspecific activation of ACTH receptor (ACTHR) (96, 97, 98). VIP/PACAP-induced adrenal steroidogenesis can also be enhanced by an indirect mechanism: indeed, both stimulate catecholamine secretion from adrenal chromaffin cells (99, 100), which in turn elicit a ß-adrenoreceptor-mediated aldosterone release (101, 102). Moreover, cortisol secretion can be raised by increasing the intraadrenal blood flow as it is stimulated by VIP and PACAP (103, 104).
Ang-II, the biologically active peptide of the RAS, and potassium ion are the major regulators of aldosterone synthesis and secretion (2). A decrease in potassium balance activates the RAS, leading to Ang-II, and then to aldosterone release. Ang-II mediates its effect on steroidogenesis via AT1 receptors (AT1R), which are coupled to phospholipases C and A2 (PLC, PLA2). It has been demonstrated that Ang-II inhibits the expression of P450c17 at the transcriptional level in ovine adrenocortical cells (105). Moreover, it augments the expression of StAR protein (106). In the rat, Ang-II enhances the transcription of AT1R and P450 aldo synthase (CYP 11B2) in vivo and in vitro (107, 108). However, Ang-II seems to inhibit AT1R expression in bovine and human fasciculata cells (109, 110). The presence of a local RAS in the adrenal cortex suggests that Ang-II can regulate aldosterone production in a paracrine fashion (111) (for review see Refs. 112, 113). Inhibitory signals contribute to maintain aldosterone homeostasis. Dopamine and somatostatin blunt Ang-II-induced aldosterone production (114, 115). The natriuretic peptides ANP and C-type natriuretic peptide (CNP), which are present in the circulation but are also expressed in the adrenal medulla, have been demonstrated to exert an inhibitory action on aldosterone release in vitro (116, 117). ANP also inhibits ACTH and Ang-II-induced cortisol production by decreasing the level of StAR expression (118). Other neuropeptides regulate the steroidogenic function of the adrenal cortex by acting both at the central and adrenal levels, as endothelin 1 (ET-1) (119, 120) and NPY (121, 122) enhance cortisol and aldosterone release.
Recent attention has been drawn to leptin as a negative regulator of the HPA axis. Acute injection of leptin in humans (123) and mice (124) counteracts fasting-induced activation of the HPA axis. This effect is proposed to be driven by a direct action of the peptide, both at the hypothalamic and adrenal levels (125). Leptin and its receptor, Ob-R, are expressed in the pituitary (126, 127) and in human, rat, and mouse adrenal glands (128, 129, 130). Moreover, the adrenal is embedded in adipose tissue, the physiological source of leptin, which acts at the transcriptional level to prevent the stress-induced stimulation of CRH and CYP17 mRNAs in the hypothalamus and adrenal, respectively (131, 132, 133). Other studies have shown opposite effects of leptin on the pituitary where CRH (known to suppress appetite and food intake) and ACTH levels are stimulated, leading to cortisol secretion (134, 135). These discrepancies may arise from anatomic and functional differences in CRH neurons in the PVN where leptin might have inhibitory effects on some and stimulatory effects on other populations of cells. Leptin is induced by GCs (136, 137), resulting in higher plasma levels in CS patients (138, 139).
The integrity of adult adrenal size is maintained by a continuous process of cell division in the ZG and centripetal migration and differentiation into fasciculata cells (140). Chronic stimulation by ACTH induces a phenotypic change of glomerulosa cells into fasciculata cells (141) whereas GCs inhibit this differentiation process namely by reducing P450scc expression (142, 143, 144); it was proposed that GCs may play a role in the functional zonation of the adrenal cortex (11). Indeed, high levels of GC (as high as in the inner adrenal cortex owing to centripetal blood flow) were shown to inhibit the 18-hydroxylation step in ACTH-treated cultures of human fetal adrenals, thus decreasing 18-OH-deoxycorticosterone (DOC) and aldosterone levels (11). In contrast to GC, ACTH can lead in vivo to hypertrophy and hyperplasia of the adrenal cortex, a process that is reversible. Paradoxically, it seems to harbor inhibitory effects on cell proliferation in vitro. A trophic effect is observed after a 2-h exposure to ACTH. This is correlated with a PKA-dependent increase of c-Jun and c-Fos expression (145, 146). After 24 h of stimulation, c-Myc expression is decreased, and inhibition of cell growth is observed (145, 147). Recent data suggest a cAMP-independent proliferation-promoting effect of ACTH (148, 149). Indeed, ACTH was shown to stimulate the mitogen-activated protein (MAP)-kinase pathway in vivo and in vitro, leading to the accumulation of c-Fos, c-Jun, and c-Myc (147, 150). Ang-II is another peptidic hormone that can also activate the MAP-kinase cascade in adrenal cells in a PKC-dependent mechanism (146, 151). In vivo, a chronic stimulation with Ang-II induces ZG hypertrophy. ET-1 also augments cell proliferation in the ZG in vitro and in vivo by interacting with its ETA receptor, which is specifically expressed in the ZG (119). Chronic treatment with VIP exerts a moderate hyperplasia of ZG in vivo (152, 153). Somatostatin exerts direct antiproliferative effects on the ZG in vivo (115). It can also antagonize the mitogenic action of Ang-II. ACTH stimulates the autocrine production of growth factors (GFs) such as insulin-like growth factor I (IGF-I), IGF-II, and transforming growth factor-ß1 (TGF-ß1), which regulate the trophic and steroidogenic functions of the adrenal cortex in vivo (11, 154). IGF-I and IGF-II have mitogenic effects. IGF-II is more highly expressed in fetal than in adult adrenals (155). In addition, it is highly expressed in hormonally active adrenocortical carcinomas but not in benign tumors, which suggests an important role in tumor acquisition or progression (156, 157). In bovine cells, IGF-I and TGF-ß1 exert opposite effects on adrenocortical function by inhibiting the expression of specific adrenal genes; IGF-I enhances the transcription level of ACTH-R, StAR, and specific steroidogenic enzymes, whereas TGF-ß1 inhibits it (158). TGF-ß1 is thought to play a role in human fetal adrenal remodeling, as it inhibits fetal zone cell proliferation and promotes apoptosis in vitro (159, 160). However, this has not been demonstrated in vivo.
| III. Primary Adrenal Cushings Syndrome (CS) |
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Primary adrenal etiologies account for 1520% of endogenous CS in adults and are secondary to unilateral tumors in 9098% of cases (1, 2, 163); in contrast, in prepubertal children, primary adrenal causes are responsible for almost 65% of CS. In adults, some case series have suggested that adenomas and carcinomas are equally responsible for adrenal CS, whereas in other series, adenomas were responsible for up to 80% of cases (165, 166). Cortisol-secreting adrenal carcinomas are 34 times more frequent than adrenal adenomas in children. For unclear reasons, adrenal tumors are more frequent in females than in males with a ratio of 4:1 for adenomas and 2:1 for carcinomas (161, 162, 163, 164).
Less than 10% of ACTH-independent CS can be secondary to bilateral
adrenal lesions, and their pathophysiology is diverse. Primary
pigmented nodular adrenocortical disease (PPNAD) or micronodular
adrenal dysplasia can be familial, associated with other tumors such as
myxomas, schwannomas, pigmented cutaneous lesions, and peripheral
endocrine tumors (Carneys complex), and linked to unknown genes on
chromosome 2 or to mutations of protein kinase A Type 1-
located on
chromosome 17 (167, 168, 169 169A ). In PPNAD, the overall size of the
adrenal gland is usually not enlarged, but is occupied by several small
black or brown nodules spread in an otherwise atrophic cortex. High
synaptophysin expression in PPNAD nodules suggests a neuroendocrine
phenotype of these cells (170). A paradoxical increase in cortisol
production is often found in these patients during Liddles
dexamethasone suppression test (171). In McCune-Albright syndrome,
activating mutations of Gs
occur in some
adrenal cells in a mosaic pattern during early embryogenesis and lead
to the formation of adrenal nodules, in which constitutive activation
of AC and the steroidogenic cascade produce increased cortisol
secretion with ACTH suppression; the internodular adrenal cortex, where
the Gs
mutation is not present, becomes
atrophic (172, 173).
ACTH-independent bilateral macronodular adrenal hyperplasia (AIMAH) is
a rare cause of CS, as it is estimated to represent less than 1% of
all endogenous cases of this syndrome (1, 2, 3, 4). In a review by Lieberman
et al. (174) in 1994, only 24 published cases had been
identified, but several other cases and series have been reported since
then (175, 176, 177, 178). AIMAH has been described by various terms, including
massive macronodular adrenocortical disease (MMAD), autonomous
macronodular adrenal hyperplasia (AMAH), ACTH-independent massive
bilateral adrenal disease (AIMBAD), and "giant" or "huge"
macronodular adrenal disease (175). The clinical syndrome becomes
evident during the patients fifth or sixth decade and has a
relatively even gender distribution when compared with Cushings
disease or unilateral adrenal tumors, which are more prevalent in
women. Most cases have been sporadic, but a few familial cases have
been reported as well (179, 180, 181, 182). An activating R201S mutation of
Gs
was found in the AIMAH tissues of a patient
without any other features of McCune-Albright syndrome (183).
| IV. Initial in Vitro Evidence of Ectopic Adrenal Membrane Hormone Receptors |
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-, adrenergic agonists. Further
studies (Table 1
- adrenergic receptors stimulating guanylate
cyclase and cGMP production was also demonstrated in rat adrenal
carcinoma 494 (188, 189).
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Other in vitro studies have further supported the functional
coupling of several, most frequently G protein-linked, membrane hormone
receptors to steroidogenesis in some human adrenocortical benign and
malignant tumors (Table 1
). Millington et al. (190)
investigated the effects of various hormones on the secretion of
steroids in a human feminizing adenocarcinoma secreting mostly
estrogens and androgens, but also some GC. AC activity was stimulated
more by PRL, human placental lactogen, LH, and FSH preparations than by
ACTH; insulin inhibited AC slightly, while TSH was without effect. In
tumor explant culture, estrone and estradiol secretion was stimulated
by PRL, insulin, and ACTH, but little by LH or GH. Androstenedione
secretion was augmented by LH, GH, PRL, and ACTH. The synthesis of
11-hydroxycorticosteroids was stimulated by LH, GH, and PRL, but very
little by ACTH. It must be stressed that hormone preparations available
at that time were not pure and that contamination was quite possible.
Matsukura et al. (191) studied AC activity in human
cortisol-secreting adrenal tissues from adenomas, adenocarcinoma, and
primary nodular hyperplasia (AIMAH), compared with normal adrenals and
bilateral hyperplasias from pituitary Cushings disease. In normal
tissues, only ACTH and PGE1 stimulated AC
activity; in most adenomas, AC activity was increased by
norepinephrine, in some by epinephrine, and in a few by TSH, LH, or
Ang-II. In a case of AIMAH, AC was stimulated by glucagon and ACTH
only. No stimulation of AC was found in adrenal carcinoma tissue.
Hirata et al. (192) demonstrated the presence of
high-affinity ß-adrenergic binding sites in two of three
cortisol-secreting adenomas, but not in the normal adrenal cortex or in
one case of aldosterone-producing adenoma; furthermore, epinephrine
stimulated cortisol secretion in cultured tumor cells from one of the
patients with an adenoma, and Katz et al. (193) studied six
human adrenal carcinomas with diversified steroidogenic activities and
compared them with the normal adrenal cortex from three individuals; AC
was stimulated by ß-adrenergic agonists in four of six tumors but not
in normal tissues. In one tumor examined for other hormone responses,
AC was also stimulated by TSH, but not by glucagon or hCG. In two
cases, membranes from metastatic adrenocortical cancer were compared
with the primary tumor and had lost stimulation of AC by epinephrine or
ACTH. Specific high-affinity ß-adrenergic binding sites were detected
only in tumors in which AC was stimulated by ß-adrenergic agonists.
In contrast, Saez et al. (194) did not find any AC
responsiveness to norepinephrine, glucagon, and TSH in crude adrenal
membranes from 11 patients with adenomas and carcinomas.
The aberrant expression of LH/hCG receptors was also previously reported in vitro in androgen-secreting adrenal adenomas (195, 196). Testosterone production was stimulated by hCG and ACTH in adrenal adenoma cells in culture, while only ACTH but not hCG was able to stimulate secretion of cortisol, testosterone, and other steroids from the adjacent normal adrenal cortex (195); binding studies performed on cell membranes from hCG-responsive adrenal adenoma demonstrated high-affinity (0.14 nM) binding capacity (198 fmol/g). A preliminary report of the presence of LH/hCG receptor in a cortisol-secreting adrenocortical carcinoma was presented recently (197).
Willenberg et al. (198) investigated the adrenal adenoma of a 62-yr-old woman who presented CS with no particular clinical characteristics; striking lymphocytic infiltration of the adenoma was identified at histology. In contrast to normal control human adrenals or other cortisol-secreting adenomas or carcinomas, immunostaining revealed CD45 and CD68-positive macrophage-like cells in this patients adenoma, and these cells are a major source of IL-1. Type I IL-1 receptor, which is not a seven-transmembrane G-coupled-receptor, was also found to be aberrantly expressed in the adenoma, by in situ hybridization and RT-PCR, but not in the normal adrenal cortex or other tumors. In cells dispersed from the adenoma, cortisol secretion was stimulated 2.6-fold by IL-1ß, but poorly by ACTH (198); in normal adrenocortical cells or other cortisol-secreting adenomas, cortisol secretion was increased by approximately 1.5-fold during incubation with IL-1ß. Since infiltration of mononuclear cells occurs in 15% of adrenal tumors, it will be of interest to further explore the prevalence of abnormal cytokine receptor expression in adrenal hyperplasias and tumors.
| V. In Vivo Demonstration of the Functionality of Ectopic or Abnormal Membrane Hormone Receptors |
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A. Food- and GIP-dependent CS
Hamet et al. (199) were the first to identify
"food-dependent" cortisol production in a 41-yr-old male patient
presenting with CS secondary to a unilateral adrenal adenoma and
periodic hormonogenesis. Plasma cortisol was consistently low in the
morning or during fasting, but increased to abnormal levels after
meals; food-induced elevations of plasma cortisol were not suppressed
by high oral doses of dexamethasone. AC activity in the resected
adrenal adenoma membrane preparation was stimulated 27% by ACTH and
62% by vasopressin, but not by FSH, glucagon, or Ang-II; the effects
of various gastrointestinal hormones were not examined in this case.
Another female patient with CS secondary to an adrenal adenoma had been
previously reported to have "persistent diurnal cortisol secretory
rhythm" (202); the low fasting plasma cortisol levels in the morning
increased during the day at the presumed, but not indicated, meal
times, suggesting that this patient also had food-dependent CS.
Two patients with bilateral AIMAH and food-dependent cortisol
production were studied in detail a few years later and allowed to
clarify the pathophysiology of this syndrome (200, 201). The first
patient, a 48-yr-old French-Canadian woman, presented with typical
symptoms of CS, which had become manifest during the previous 23 yr
(200). Initial investigation revealed low plasma cortisol
levels, fasting in the morning, and higher levels during the
day, whereas plasma ACTH was always suppressed. The suspicion that
cortisol production was regulated by a gastrointestinal hormone came
from the observation that plasma cortisol was stimulated by oral
administration of glucose or by lipid-rich or protein-rich meals, but
not by intravenous glucose. In addition, somatostatin pretreatment
inhibited the cortisol-stimulatory effect of oral glucose. A review of
the various secretagogues of gastrointestinal hormones indicated that
only GIP and the glucagon-like peptides (GLPs) were stimulated
significantly by oral glucose and lipids, and to a lesser extent by
proteins. Plasma cortisol levels were correlated with plasma GIP
concentrations during the various test meals. In vivo GIP
infusion, to reproduce physiological postprandial concentrations,
augmented cortisol production in the patient, but not in four normal
controls. In the patient, plasma cortisol was stimulated by the
administration of ACTH but not by CRH, glucagon, insulin-induced
hypoglycemia, pentagastrin, or AVP. The presence of GIP receptors
(GIPRs) in adrenal tissues was supported by adrenal imaging after the
injection of [123I]-GIP in vivo
(200). The incubation of dispersed adrenal cells in vitro
confirmed GIP-mediated cortisol secretion in the patients cells,
whereas no cortisol response to GIP was found in normal adult or fetal
adrenal cells or in other cortisol- or aldosterone-secreting adenomas
(200); there was no stimulation of cortisol production in the
patients adrenal cells after in vitro incubation with
secretin, CCK, VIP, substance P, bombesin, calcitonin gene-related
peptide, glucagon, vasopressin, ANP, CRH, TRH, GHRH, neurotensin, or
neurokinin A. It was thus concluded that food-dependent cortisol
secretion resulted from the abnormal responsiveness of adrenal cells to
the physiological secretion of GIP; "illicit" or ectopic GIPR
expression on adrenal cells (Figs. 1
and 2
) presumably were the basis for
this new etiology of CS (200).
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Food- or GIP-dependent CS has now been identified in 13 patients with
AIMAH (139, 200, 201, 205, 206, 207, 208) and in seven with unilateral adenoma
(199, 205 208A, 213), as summarized in Table 2
. At pathological examination, no
distinctive features were reported, compared with non-GIP-dependent
cortisol-secreting adenomas or bilateral macronodular hyperplasia,
except in one case (207). This patient was described in a preliminary
report to have facial pigmented spots, a blue nevus on one leg,
lipofuscin pigments in bilateral adrenal macronodules, and a
periadrenal schwannoma suggestive of Carneys complex without any
family history; a full description has not yet been published, but
in vitro studies clearly confirmed GIP-induced stimulation
of cortisol secretion by adrenal cells (205). In two cases of AIMAH,
the patient initially presented with a unilateral lesion and developed
contralateral enlargement only later in time (206 208A ). Except for
three patients [the first patient described with food-dependent CS
but not proven to be GIP-dependent (199) and two recent ones with AIMAH
(GIPR overexpression not yet confirmed)], all other patients are
females; adrenal CS is more frequent in females (161), but it remains
to be seen whether an even higher female frequency will be found in
GIP-dependent CS and what molecular mechanism underlies this sex
distribution. Average age at the time of diagnosis may be somewhat
greater in patients with AIMAH than in patients with unilateral adrenal
adenoma (Table 2
) (174, 175); the youngest patient with a unilateral
adenoma was only 15 yr old. In GIP-dependent CS, chronic GIP-induced
hypercortisolism eventually leads to suppression of CRH and ACTH; this
suppression, coupled with low GIP levels in the fasting state, is
responsible for the decreased plasma cortisol levels, which can be
accompanied by symptoms of relative cortisol insufficiency (201, 209).
However, in certain patients (Table 2
), fasting plasma cortisol levels
were not particularly low, indicating that GIP-dependent CS should not
be excluded without performing a test meal (139, 206); this finding
could indicate that subpopulations of adrenal cells in the tumor or
hyperplasia have lost their GIP dependency and are secreting cortisol
under different mechanisms, or that more than one abnormal receptor
regulating cortisol production are expressed in these cells. In one
patient with food-dependent AIMAH but in whom fasting plasma cortisol
was relatively elevated, Pralong et al. (139) reported that,
in addition to GIP, leptin also aberrantly stimulated cortisol
secretion in dispersed adrenal cells; thus, the potential presence of
more than one abnormal receptor may modify the phenotypic appearance.
The potential presence of ectopic GLP-1 receptors has been excluded to
date by the lack of stimulation of cortisol production after GLP-1
administration, either in vivo or in vitro (139, 206, 210). In one patient with GIP-dependent AIMAH, plasma ACTH and
cortisol responses to CRH were still preserved, presumably because the
intermittent food-dependent stimulation of cortisol had not yet
completely suppressed the HPA axis (208). In a female patient with
hirsutism and a unilateral adenoma, both adrenal androgens and cortisol
were found to be stimulated by food intake in vivo and GIP
in vitro (213); hypercortisolism was modest and ACTH was not
fully suppressed.
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De Herder et al. (209) used in situ hybribization
to demonstrate abundant GIPR mRNA in adrenal adenoma cells from their
patient with GIP-dependent CS; this signal was not present in the
adenoma from a patient with non-food-dependent CS, but was not examined
in the normal adrenal cortex in this initial study. Using RT-PCR
amplification, N'Diaye et al. (219) demonstrated pronounced
adrenal GIPR overexpression in adrenal adenoma or hyperplastic tissues
from GIP-dependent CS compared with the normal human pancreas, normal
adult or fetal adrenal cortex, or non-GIP-dependent adrenal CS tissues.
A small amount of GIPR mRNA was detected in normal fetal and adult
adrenal tissues after at least 35 cycles of amplification and
hybridization with the labeled cDNA but was not coupled efficiently to
steroidogenesis. Sequence analysis of the full-length cDNA of normal
and GIP-dependent adrenal tissues revealed no mutation of GIPR in the
affected adrenal tissues (219); similar proportions of isoforms lacking
exons 4 and 9 were identified in normal and GIP-dependent adrenals.
Chabre et al. (210) confirmed the presence of the same
overexpressed GIPR isoforms in a GIP-dependent adenoma by RT-PCR and
sequencing; no GIPR bands could be detected in the atrophic adrenal
cortex adjacent to the tumor or in normal adult adrenals, but only
ethidium bromide staining was used. The ACTHR was found to be expressed
at a lower level in GIP-dependent adenoma compared with normal tissues
(210); this may be secondary to the chronic suppression of endogenous
ACTH, which is known to up-regulate ACTHR expression (36, 37). If the
relative suppression of ACTHR in GIP-dependent adrenal tissues is
confirmed in further studies, this would indicate that GIP cannot
substitute for ACTH in inducing the expression of ACTHR; it must
be noted, however, that plasma GIP levels are only elevated transiently
postprandially, which is different from conditions where ACTH is
elevated chronically. GIPR overexpression was confirmed in other cases
(Table 2
) of GIP-dependent adrenal macronodular hyperplasias (205, 206, 208 208A ) and adenomas (205 208A, 210, 213) and was not demonstrated
in non-GIP-dependent CS adrenal tissues (205, 210, 213, 219) or the
human adrenocortical carcinoma cell line H295 (211). GIPR
overexpression was detected, even in the early stages of adrenal
hyperplasia (206). The small amount of GIPR mRNA sometimes found in
normal fetal or adult adrenal tissues after amplification was not
efficiently coupled to steroidogenesis (219) and may reflect a low
number of GIPR in endothelial cells (214) rather than in adrenocortical
cells. Thus, the concept of functional ectopic receptors remains valid
in explaining the pathophysiology of GIP-dependent CS (Figs. 1
and 2
).
It has been reported that the in vitro cortisol-stimulating effects of GIP are coupled to an increase of cAMP, but not of IP3 production (205, 210). In studying GIP-dependent adrenal cells in primary culture, GIPR down-regulation by its own ligand has been demonstrated, as assessed by the induction of steroidogenic enzyme expression, cortisol secretion, or GIPR mRNA levels by in situ hybridization and RT-PCR studies (205, 220). By stimulating steroidogenic enzyme activity, ACTH pretreatment of cells increased the GIP-induced cortisol response but did not appear to modify GIPR expression directly (205).
Stimulation of thymidine incorporation into newly synthesized DNA by GIP was observed in primary cultures of adrenal cells from GIP-dependent CS, but not in normal cells (210). Activation of p42-p44 MAP kinases was observed after treatment of pathological cells with GIP (210). Depending on the cell culture conditions used, ACTH can be shown to inhibit or stimulate markers of cell proliferation in adrenal cells. In the studies by Lebrethon et al. (205), under conditions where ACTH inhibited thymidine incorporation in normal and GIP-dependent adrenal cells, GIP was also found to suppress DNA synthesis only in GIP-dependent, and not in normal adrenal cells. Such results suggest that GIP is possibly capable of regulating cell proliferation, in addition to steroidogenesis, in these tissues; however, cell growth stimulation by GIP has not yet been clearly demonstrated.
It should be stressed that food-induced cortisol secretion has been
reported in some non-GIP-dependent CS. Bercovici et al.
(221) described a patient with pituitary Cushings disease in whom
ACTH and cortisol were increased strikingly after mixed meals. ACTH
secretion was stimulated by protein-rich meals, but not by oral glucose
or lipid-rich meals. Intravenous infusion of amino acids was capable of
inducing this response, while octreotide administration did not modify
urinary cortisol levels. It was concluded that the pituitary
corticotroph adenoma of this patient retained the capacity that normal
corticotroph cells have to enhance their release of ACTH after protein
ingestion. It has been shown very clearly that, in normal individuals,
mixed meals produce an increase in ACTH release and in plasma cortisol
levels; this is more evident at lunchtime than after breakfast, when
the diurnal peak of ACTH and cortisol may mask the response (222, 223, 224).
This stimulation is of hypothalamic-pituitary origin and is abolished
by dexamethasone administration (225). It is believed that the effect
may be secondary to the heightened serotonin production and related to
tryptophan content in the meal (224).
-Adrenergic agonists can also
increase postprandial stimulation of ACTH (226).
B. Vasopressin-responsive CS
A large proportion of pituitary corticotroph adenomas have been
shown to augment their ACTH release after LVP administration, resulting
in increased plasma cortisol levels (227, 228). In contrast, in adrenal
CS, where ACTH is suppressed, it is expected that plasma cortisol
should not increase after LVP administration (229). However, abnormal
adrenal stimulation of cortisol secretion in response to exogenous AVP
or LVP administration has been described in canine (230) and human
ACTH-independent CS, secondary to unilateral adrenal adenomas,
carcinomas, or AIMAH (Table 3
).
|
Horiba et al. (234) reported two male Japanese patients with bilateral macronodular adrenal hyperplasia and clinical CS in whom im injection of 10 IU LVP increased plasma cortisol 2.3- to 2.6-fold, while plasma ACTH remained undetectable; there were no ACTH or cortisol responses to CRH or dexamethasone. Upon pathological examination, the glands were replaced by macronodules composed of compact and clear cells, but there were some regions of cortical internodular atrophy. In dispersed adrenal cells from both patients, LVP stimulated cortisol secretion (2.8- to 3.2-fold) more efficiently than ACTH. In seven other patients with CS and unilateral adenoma, LVP injection resulted in small increases of plasma cortisol, varying between 9.8 and 25.3%. In four normal subjects pretreated with 2 mg dexamethasone at bedtime and 0.5 mg on the morning of the test, LVP injection elevated plasma cortisol 1.6- to 1.8-fold (up to 45 nmol/liter from basal levels of 20.9 nmol/liter). An exaggerated 2.6-fold rise in plasma cortisol after 10 IU of LVP was also reported in a patient with a unilateral cortisol-secreting adenoma and mild ACTH-independent CS (235). Intracellular calcium flux in dispersed tumor cells was stimulated by AVP and inhibited by a V1-AVPR antagonist. Using RT-PCR amplification, the V1-AVPR signal was stronger in the cortisol-secreting tumor than in the normal gland; there was a faint V2-AVPR signal in normal and tumoral adrenal tissues, and no V3-AVPR in either.
A 36-yr-old female American patient with CS and AIMAH presented an unusual association with orthostatic hypotension (80). Exogenous AVP, but not desmopressin, triggered large elevations of plasma cortisol (3.4-fold) and aldosterone (67-fold) levels. During upright posture and hypotension, cortisol and aldosterone secretion increased, despite the suppression of ACTH and renin levels. AVP, which normally rises during upright posture and even further in orthostatic hypotension, remained below the limit of assay detection, until the correction of hypercortisolism. Under dexamethasone suppression, plasma cortisol, aldosterone, and androgens were elevated by exogenous AVP in the patient, but not in the controls. Cells freshly dispersed from the diffuse adrenal hyperplasia displayed higher cortisol stimulation (4.2-fold) during incubation with AVP than normal adrenal cells (1.3-fold); the cortisol response was mediated by V1-AVPR, as shown by the effects of V1 antagonists and the lack of effect of V2 agonists. The presence of V1-AVPR was supported by binding studies, intracellular Ca2+ flux studies, and RT-PCR amplification of mRNA for all three AVPR. The binding studies revealed a similar V1-AVPR affinity (2.63 nM) in AIMAH adrenal cells, compared with membranes from human glomerulosa-rich normal adrenal cells or myometrium (236). The ED50 of AVP on [Ca2+]i was similar in the adrenal cells of the patient (0.9 nM) compared with glomerulosa-rich cells (1.4 nM) from normal adrenals (76). Interestingly, CRH administration stimulated cortisol in vivo but not in vitro without any stimulation of ACTH; it is possible that CRH increased the adrenal production of vasopressin (68) and cortisol in a paracrine manner. Alteration of the V1-receptor-effector system was not limited to the adrenal tissues of this patient, as there was also an abnormal, prolonged vascular vasoconstrictive response to AVP, compared with the arterioles of normal or hypertensive subjects. The persistence of decreased stimulation of plasma vasopressin and endothelin levels during postural hypotension, several months after correction of the hypercortisolism, also raised the possibility of an exaggerated V1-AVPR signal at the hypothalamic level in this patient. The causal relationship between abnormal V1-AVPR-mediated-responses and postural hypotension remains uncertain (80). Another male Japanese patient with AIMAH and CS was found to have a 1.8-fold increase in plasma cortisol after LVP injection (237); food intake, GIP infusion, octreotide, and CRH were without effects. Removal of the large bilateral macronodular adrenals showed no areas of internodular atrophy; LVP stimulated cortisol production in cells freshly dispersed from a macronodule. Stimulation of plasma cortisol by administration of 0.2 IU AVP was noted in a Japanese man with AIMAH and coincident multiple adenomatous polyps and colon cancer (238); a point mutation of the APC gene was revealed in the colon cancer but not in the adrenal nodules.
In a retrospective study of 26 patients with CS secondary to unilateral
cortisol-secreting tumors, Arnaldi et al. (79) observed an
increase of plasma cortisol greater than 30 ng/ml after LVP testing in
27% of cases (five adenomas and two carcinomas). Quantitative RT-PCR
assay of V1-AVPR showed that the levels of message were similar in 20
cortisol-secreting adenomas, compared with three normal adult adrenals;
the levels were lower in 19 adrenocortical carcinomas, but there was a
large overlap with adrenal adenomas. The normal adrenal glands and the
majority of tumors also expressed low amounts of V2-AVPR, but no
V3-AVPR. Only six of the patients for whom adrenal tumor material was
available had undergone LVP testing; responders had somewhat higher
V1-AVPR concentrations in their tumors than nonresponders, but the
levels were not higher than in normal adrenal tissues. In one patient
with an in vivo cortisol response (
1.6-fold) to LVP, the
AVP-induced cortisol secretion (2-fold) of perifused adrenal cells was
inhibited by V1-AVPR antagonists.
The demonstration of an exaggerated cortisol response to pharmacological levels of exogenous vasopressin does not constitute direct evidence that fluctuations of endogenous AVP levels are the main regulator of steroidogenesis in these patients. This was illustrated in a male patient with AIMAH who was shown to have increased plasma cortisol in response to upright posture and administration of 10 IU AVP (86); however, the modulation of endogenous AVP levels by water dilution or hypertonic saline infusion did not modify plasma cortisol levels. In addition, in vivo administration of a V1-AVPR antagonist inhibited the response of cortisol to exogenous AVP, but not to upright posture. In fact, this patient was found to have ectopic ß-adrenergic receptors (see Section V.C.) in his adrenal tissues; it is believed that pharmacological AVP levels stimulated catecholamine release, including from the adrenal medulla (68), and then mediated cortisol release in this case. Further support comes from the fact that there was no evidence of V1-AVPR in his adrenal tissues (N. NDiaye and A. Lacroix, unpublished observation).
Daidoh et al. (239) studied a 49-yr-old man with very large bilateral AIMAH and severe CS; intravenous injection of small amounts of AVP (0.3 IU) increased plasma cortisol 3.7-fold without any detectable rise in ACTH. Similarly, insulin-induced hypoglycemia elevated plasma AVP and cortisol without any increase in plasma ACTH; catecholamine effects were not studied however. Upright posture augmented plasma AVP and cortisol. Oral administration of the V1-AVPR antagonist OPC-21268 for 8 days decreased urinary free cortisol levels, but potential spontaneous fluctuations of cortisol secretion were not evaluated for long periods. It was further shown, in dispersed adrenal cells, that AVP stimulated cortisol secretion in AIMAH cells but not in normal control cells, and that this effect was inhibited by OPC-21268; GIP was without effects on AIMAH cells, but catecholamine and insulin were not tested directly. We recently studied a 50-yr-old American woman with CS and AIMAH in whom plasma cortisol was stimulated by upright posture (1.7-fold) and exogenous AVP (3.4-fold), but not by dDVAP (240). In this patient, we were able to demonstrate that plasma cortisol was inhibited by water loading (24% decrease), and elevated during hypertonic saline infusion (1.7-fold). This patient was also found to have abnormal responses to ß-adrenergic receptor agonists (see Section V.C.), in addition to the abnormal V1-AVPR response in her adrenals. These last two cases represent the first demonstrations of fluctuations in plasma cortisol levels in parallel with small physiological changes in endogenous vasopressin levels. All the previously reported cases of cortisol stimulation by lysine- (231, 232, 233, 234, 235, 237) or arginine-vasopressin (80) were related to exogenous pharmacological amounts. In these last two patients, as in another patient (80), plasma vasopressin was found to be suppressed to undetectable levels basally and showed only a very modest increase upon potent physiological stimulation. This may be due to the suppressive effects of hypercortisolism on vasopressin gene expression (241). It has also been postulated that abnormal V1-AVPR may modify vasopressin production via a short loop regulation mechanism in hypothalamic nuclei (80).
An abnormal increase of plasma cortisol in response to vasopressin administration was also noted in patients with preclinical bilateral macronodular adrenal hyperplasia (242). Recently, an exaggerated plasma cortisol response to LVP was seen in a 67-yr-old woman with CS and bilateral macronodular adrenal hyperplasia, whose brother had died after bilateral adrenalectomy for CS and AIMAH (182); the precise nature of the abnormal hormone receptor implicated is unknown, but this constitutes the first demonstration of abnormal hormone responsiveness in familial AIMAH.
Since V1-AVPR are present in the normal adrenal cortex and modulate modest effects of vasopressin on steroidogenesis, the exaggerated steroidogenic responses to vasopressin in these patients would be secondary to the abnormal function of an "eutopic" receptor-effector system, rather than to the presence of an ectopic receptor. V1-AVPR mRNA levels were found to be expressed either at higher (235) or similar (79, 80) levels, compared with normal control adrenal tissues. The binding affinity and dose response of intracellular calcium flux for V1-AVPR noted in the adrenal tissues of a patient with AIMAH (80) were not different from those reported in other normal tissues. Thus, no evidence of ectopic receptor or gross overexpression of the eutopic V1-AVPR has been presented to date; the molecular mechanisms leading to the abnormal response of V1-AVPR or its effector system, which would increase the response to AVP, remain to be elucidated.
Recently, V3-AVPR were shown to be expressed ectopically in a series of bronchial carcinoids secreting ACTH (243). A large proportion of patients with Cushings disease, but not normal individuals, secrete ACTH in response to DDAVP (244, 245). V3-AVPR were found to be overexpressed in corticotroph adenomas (229); as DDAVP can also bind in part to V3-AVPR, this may explain the effects of DDAVP on ACTH release in Cushings disease. Thus, stimulation of cortisol levels after vasopressin administration in CS cannot directly distinguish between pituitary corticotroph adenoma, ACTH-independent primary adrenal tumor or hyperplasia, or relatively well differentiated carcinoid tumors producing ACTH.
C. Catecholamine-dependent CS
Catecholamines are known to modulate HPA activity. Activation of
1-adrenoreceptors in the PVN leads to CRH
release with increased plasma levels of ACTH and cortisol (14).
Administration of ß1- or
ß2-adrenergic agonists or antagonists has no
effect on ACTH or cortisol secretion (246). Peripherally administered
1-adrenoreceptor agonists fail to activate the
HPA, as the blood-brain barrier prevents their access to the PVN.
Direct adrenal stimulatory or inhibitory effects of catecholamines on
GC or mineralocorticoid secretion have been noted in several species,
but are limited to aldosterone secretion in humans, where cortisol
secretion is unaffected (11).
As discussed in Section IV, the abnormal presence of ß-
adrenergic receptors or the activation of AC activity by
catecholamines has been reported in vitro in several cases
of human adrenal tumors associated with CS (191, 192, 193); no evidence of
such receptors has been found in the normal adrenal cortex. However,
the clinical expression of this abnormality was appreciated only
recently in two patients. A 56-yr-old French-Canadian man with AIMAH
and CS (86) was shown to have ACTH-independent overproduction of
cortisol and aldosterone during elevations of endogenous catecholamines
level (upright posture, insulin-induced hypoglycemia, and EKG stress
test). Augmented plasma cortisol during upright posture was decreased
after pretreatment with the ß-adrenergic antagonist, propranolol; in
contrast, this did not occur after inhibition of the RAS system with
captopril or losartan, or of AVP with a V1-AVPR antagonist.
Isoproterenol infusion stimulated cortisol (2.1-fold) and aldosterone
(2.2-fold) secretion in the patient, but not in normal subjects, in
whom ACTH had been suppressed by dexamethasone. Plasma cortisol was not
influenced by mixed meals, or administration of TRH, GnRH, glucagon, or
cisapride; as discussed previously, a late increase of cortisol after
AVP administration was believed to result from stimulation of release
of adrenomedullary catecholamines. High-affinity binding sites
compatible with ß1-adrenergic receptor
(ß1-AR) or ß2-AR were
found in the adrenal tissues of the patient, but not in the controls.
They were efficiently coupled to steroidogenesis (Fig. 1
), as shown by
AC stimulation with isoproterenol in vitro and
catecholamine-induced steroidogenesis in vivo (86). Further
molecular studies are needed to properly characterize the
ß-adrenergic receptor subtype expressed in hyperplastic adrenal
tissues and to determine whether or not it is mutated.
Another 50-yr-old American woman with CS and AIMAH (240) was found to have abnormal responses to catecholamines in addition to an exaggerated response to AVP (described previously in Section V.B.). In this patient, plasma cortisol had risen after upright posture (1.7-fold) and exogenous AVP (3.4-fold), but also after insulin-induced hypoglycemia (2.7-fold), while ACTH remained suppressed. Infusion of isoproterenol for 30 min increased plasma cortisol from 323 to 630 nmol/liter, which returned rapidly to baseline when the infusion was discontinued. Pretreatment of the patient with the angiotensin receptor type-1 antagonist losartan did not prevent the elevation of plasma cortisol during upright posture. There were no increases of plasma cortisol after mixed meals, GnRH, TRH, glucagon, or cisapride. It was concluded that cortisol secretion was mediated by the abnormal presence and function of ß-adrenergic and V1-AVPR, and medical therapy with the ß-blocker propranolol was proposed to the patient; she did not tolerate this medication well and elected to undergo surgery in her home city (tissues not available).
D. LH-dependent CS
The LH/hCG receptor (LH/hCGR) normally activates AC and PLC to
stimulate gonadal steroidogenesis (247). The receptor is mainly
expressed in gonadal tissues, but also in other tissues, including the
uterus, fallopian tubes, placenta, brain, hypothalamus, and prostate
(248); recently, the presence of LH/hCGR was identified in the zona
reticularis of the human adrenal (249) by immunohistochemistry and
in situ hybridization. hCG stimulates DHEAS secretion in
human fetal adrenal cells (250).
A 63-yr-old French-Canadian woman was studied for CS and AIMAH (251).
Retrospectively, she described having gained between 1822 kg during
each of four full-term pregnancies, with Cushingoid fat distribution,
but without high blood pressure, purple skin striae, or hirsutism. Her
weight returned rapidly to baseline after delivery with symptoms of
lack of appetite, nausea, and fatigue, which subsided within 23
months. Chronic hypercortisolism became clinically manifest only 10 yr
after menopause (Fig. 3
). Cortisol
production was increased by the in vivo administration of
GnRH, hCG, and recombinant human LH (hLH). Plasma free testosterone and
estradiol were also augmented by hLH administration. Abnormal
stimulation of cortisol, free testosterone, and DHEAS production was
also evoked in this patient by oral intake of cisapride and
metoclopramide, two 5-HT4R agonists (251).
Administration of the long-acting GnRH analog leuprolide
acetate initially increased LH and FSH secretion, which was paralleled
by a rise in cortisol secretion; however, this was followed within 10
days by suppression of endogenous LH and FSH levels and normalization
of cortisol production. Stimulation of cortisol by hCG and recombinant
hLH, but not by FSH, suggests that a functional adrenocortical LH/hCGR
was coupled to steroidogenesis (Fig. 3
); the lack of stimulation by
GnRH, when LH levels were suppressed by chronic administration of
leuprolide acetate, excludes an adrenal GnRH receptor. Studies of
normal adult controls did not indicate any coupling of LH/hCGR to
adrenal synthesis of cortisol or DHEAS. Abnormal stimulation of plasma
cortisol after GnRH and LH administration was also found in one woman
with bilateral macronodular adrenal hyperplasia and normal urinary
cortisol levels, which did not suppress normally with dexamethasone
(242). This suggests that diverse ectopic hormone receptors can be
present in preclinical bilateral macronodular adrenal hyperplasia.
|
Transient corticotropin-independent CS during pregnancy with complete resolution after spontaneous abortion or delivery was described in two patients with mild bilateral adrenal hyperplasia (260, 261) or unknown adrenal pathology (262). A paradoxical increase in cortisol excretion during dexamethasone administration in pregnancy completely returned to normal after delivery (261). One patient developed severe biochemical and clinical evidence of CS during each of three pregnancies, and ACTH or cortisol levels were not stimulated by vasopressin administration (260); there was a transient period of hypocortisolism after each delivery, followed by complete clinical regression. In one case, short-term hCG administration elevated urinary 17-hydroxycorticosteroid levels, while sequential estrogen and progestogen administration had no effect (262).
It is thus possible that some of these patients with transient CS during pregnancy, or in whom hypercortisolism increased during pregnancy, also expressed ectopic LH receptors in their adrenal adenomas or in their adrenal cortex. Specific testing of the regulation of steroidogenesis with LH or estrogens in future cases of transient CS during pregnancy will help in elucidating the pathophysiology. It must be pointed out that spontaneous remission of CS after delivery has also been reported in a patient with ACTH-dependent CS of probable pituitary origin (263); the mechanisms involved in this regression have not been elucidated.
E. LH-dependent adrenal androgen-secreting tumors
Although the regulation of cortisol secretion by LH in adrenal CS
was demonstrated only recently in vivo, there have been
several reports indicating that the regulation of steroidogenesis in
some rare, pure, androgen-secreting tumors was stimulated by hCG or
GnRH (195, 264, 265). As plasma LH was found to be relatively
suppressed in some of these patients, the role of endogenous LH in
maintaining androgen production may be uncertain. In some cases,
suppression of endogenous LH levels by administration of estrogens
(266, 267) or by ACTH stimulation of GC (268) inhibited androgen
production. In other cases, estrogens were unsuccessful in depressing
androgen production (264, 269, 270). It has been suggested that gonadal
cells localized in the adrenals could explain this phenomenon; however,
clear evidence of adrenal origin of the tumors was identified in
certain cases (195).
F. Serotonin-responsive CS
5-HT is produced by intraadrenal mast cells in humans and can
regulate corticosteroid production via a paracrine mechanism (87, 271);
these effects are mediated by the 5-HT4R subtype,
which is expressed mainly in adrenal ZG but also in zona fasciculata
cells (89, 91). 5-HT4R agonists are potent
stimulators of aldosterone secretion in humans; they are weak
stimulators of cortisol secretion by human adrenocortical cells
in vitro, but not of plasma cortisol in normal subjects
(87).
In the patient with LH-dependent CS (251), cisapride and metoclopramide, two 5-HT4R agonists, produced 4.8- and 2.6-fold peak elevations, respectively, in plasma cortisol 120 min after their oral administration. Plasma corticotropin levels remained undetectable during cisapride and metoclopramide testing. Stimulation of plasma cortisol in this patient after treatment with cisapride and metoclopramide was proportional to their respective affinity for 5-HT4R (87); no such response to cisapride was found in five other patients with bilateral adrenal hyperplasia, 11 with unilateral adenoma, and one with carcinoma and CS (240). A patient with CS and AIMAH was found to increase plasma cortisol in response to cisapride as well as to LVP and CRH, despite suppression of ACTH (272). Recent observations in patients with bilateral macronodular adrenal hyperplasia and preclinical hypercortisolism also documented marked stimulation of cortisol secretion upon cisapride administration (242).
The exaggerated cortisol responses to cisapride in these patients could be secondary to the increased zona fasciculata expression or abnormal function of an "eutopic" 5-HT4R-effector system, rather than to the presence of an ectopic receptor. The presence of a 5-HT4R has been detected by RT-PCR in the adrenal tissues of one of these patients and was similar to that found in normal adrenal cortex; however, full receptor sequencing and adrenal zone distribution have not been performed (272).
G. Steroid-responsive CS
Caticha et al. (273) described a 33-yr-old woman who
developed transient and reversible clinical and biochemical signs of
ACTH-independent CS during three pregnancies and during intake of oral
contraceptives. Her adrenal histology was described as being compatible
with primary nodular dysplasia, but there were no comments on
pigmentation of her adrenal nodules; there was also no family history
of adrenal disease and no other features of Carneys complex.
Paradoxical increases in cortisol production were noted during oral
dexamethasone suppression tests. Dose-responsive stimulation of
cortisol secretion occurred after bilateral adrenalectomy when the
cells were exposed to estradiol; the in vitro addition of
dexamethasone was not reported, nor were the effects of antiestrogens.
Paradoxical increases in plasma cortisol and urinary free cortisol were observed during the last 2 days of classical Liddles 4-day low- and high-dose oral dexamethasone tests in patients with PPNAD with or without Carneys complex (171). We found no evidence of ectopic membrane hormone receptors in two patients with PPNAD, who showed an increase in cortisol secretion during prolonged dexamethasone administration; GC receptors appeared to be highly expressed by immunohistochemistry in PPNAD micronodules, compared with the adjacent internodular atrophic adrenal or to the normal control adrenal cortex (274). Similar paradoxical elevations of cortisol production during dexamethasone have been reported in several cases of CS during pregnancy (258, 261).
H. Other abnormal hormone responses in adrenal CS
Hashimoto et al. (275) described a 51-yr-old male with
large bilateral AIMAH in whom plasma cortisol increased during
insulin-induced hypoglycemia, while ACTH, measured by RIA, remained at
undetectable levels (<10 pg/ml); in vitro, dispersed
adrenal cells stimulated cortisol secretion with ACTH, but not with
insulin, catecholamines, vasopressin, or Ang-II. A very similar patient
with AIMAH studied by the same group (232) also displayed elevated
plasma cortisol during insulin-induced hypoglycemia and combined
LVP-CRH tests while plasma ACTH remained undetectable; in
vitro studies were not performed in this case. It remained unclear
whether insulin itself, a factor increased during hypoglycemia, or
subdetectable rises in plasma ACTH were responsible for the regulation
of cortisol secretion in these cases.
Leptin synthesis is stimulated by GC (136), and leptin receptors are expressed in normal adrenals as well as in adrenocortical adenomas and carcinomas (128, 276). Plasma leptin has been found to be elevated in patients with CS. The leptin receptor is expressed in the adrenal cortex, where leptin normally inhibits cortisol secretion. Leptin negatively regulates the HPA, both at the pituitary level, where it suppresses CRH secretion, and the adrenal level, where it depresses steroidogenesis (124, 130, 276). Pralong et al. (139) recently reported a 36-yr-old woman with AIMAH and CS in whom a mixed meal heightened plasma cortisol levels, and this effect was decreased by octreotide pretreatment. GIP was not infused, but GIP stimulated cortisol secretion in vitro. Leptin (single dose of 100 nM) increased cortisol secretion in vitro, whereas in normal adrenal tissues, it normally suppresses this parameter. Plasma leptin levels were elevated in this patient with CS but did not increase after meals. GIPR or leptin receptor were not measured directly. Thus, this case raises the possibility of paradoxical stimulation of steroidogenesis by leptin in some cases of AIMAH, but more detailed studies are required in other similar cases to confirm this possibility.
| VI. Investigation Strategy |
|---|
|
|
|---|
B. Further characterization of abnormal hormone receptors
After initial screening, other tests can be performed to confirm
the responses or to elucidate which hormone is implicated (Fig. 4
). For example, if cortisol stimulation
by upright posture is found, the inverse effect, i.e.,
suppression by assuming a supine posture after ambulation, is verified.
The respective contributions of vasopressin, catecholamines, and Ang-II
or ANP modifications need to be distinguished. An exaggerated cortisol
response to pharmacological levels of exogenous vasopressin is followed
by evaluation of whether physiological fluctuations of endogenous
vasopressin would modify plasma cortisol levels. An increase of plasma
vasopressin during an upright posture test should parallel the
elevation of plasma cortisol levels. Endogenous plasma AVP levels can
be modulated by a 20 cc/kg water load, followed by infusion of NaCl 3%
at 0.1 cc/kg/min for 120 min. The expected result would be an initial
suppression of AVP and cortisol during water loading, followed by an
increase of AVP and cortisol levels. To determine whether the
vasopressin receptor involved in this response is a V1, V2, or V3
receptor type, 2.5 µg desmopressin, a preferential V2 receptor
agonist, is administered subcutaneously (80, 86); the absence of a
response to desmopressin would suggest a V1 or V1b/V3 receptor-mediated
response. Pretreatment with a specific oral V1 receptor antagonist (SR
49049) has been used to demonstrate in vivo the involvement
of the V1 receptor in this response (86). In case of no response to
exogenous AVP, the role of Ang-II is assessed by repeating the posture
test after administration of an AT1R antagonist or by direct infusion
of Ang-II. If a catecholamine response is suspected, endogenous
catecholamine stimulation is produced by insulin-induced hypoglycemia,
and, if positive, by isoproterenol infusion (86). An attempt to block
the response and to treat the patient with a ß-blocker would be
conducted if the stimulation of cortisol production is reproduced.
|
Stimulation of cortisol production after GnRH administration could result from the abnormal adrenocortical presence of receptors for LH/hCG, FSH, or GnRH itself. The cortisol response after the administration on different days, of hCG 10,000 U im, purified human FSH 150300 U im, and recombinant LH 300 U iv can be compared (251). A response to GnRH coupled to an absence of response to FSH, LH, and hCG would suggest an ectopic GnRH receptor; various analogs and antagonists of this receptor are available for testing the hypothesis. In addition, the response to an acute dose of GnRH should persist despite the suppression of endogenous gonadotropins by the administration of supraphysiological doses of gonadal steroids or the use of long-acting GnRH analogs. In the presence of an ectopic LH/hCGR, a response to exogenous hCG or LH, but not to exogenous FSH, should be evident; the response to acute GnRH administration should disappear when the LH response is abolished by exogenous gonadal steroids or after the chronic administration of long-acting GnRH analogs. Therapy with long-acting GnRH analogs should produce eventual suppression of the endogenous LH ligand and normalize cortisol production, as demonstrated recently by our group in one such patient (251). In the presence of an ectopic FSH receptor, there should be no response to hCG or LH, but cortisol production should be increased after the administration of purified FSH. Here again, long-acting GnRH analogs should suppress the biologically active ligand and correct the hypercortisolism.
Stimulation of cortisol synthesis after TRH administration has not yet been reported. However, AC stimulation by TSH has been demonstrated in adrenocortical adenomas in vitro (9). Thus, a response to TRH would suggest the possibility of an ectopic receptor either for TSH, TRH, or PRL. The PRL receptor does not belong to the family of G-coupled seven-transmembrane receptors, which could mimic the ACTHR and activate AC. However, adrenocortical stimulation by PRL has been described in vitro (190), and the presence of this receptor in adrenal tumors has been confirmed (278). Elevation of endogenous PRL levels after a chlorpromazine test and its inhibition by a bromocriptine test would easily clarify the role of endogenous PRL. The potential presence of an ectopic TSH receptor would be assessed directly by the administration of purified human TSH and by inhibiting endogenous TSH production with exogenous T4. The lack of an ectopic TRH receptor would be confirmed by disappearance of the cortisol response when the response of TSH to TRH has been suppressed by T4 administration.
The in vitro response of AC to glucagon has been demonstrated previously in a cortisol-secreting adenoma (190), but a clinical case has not yet been reported. If a response to 1 mg of exogenous glucagon is found, it would be necessary to show that fluctuations of endogenous glucagon levels during insulin-induced hypoglycemia, fasting, or oral administration of glucose correlate well with fluctuations of cortisol levels.
The oral administration of 10 mg cisapride, a 5-HT4R agonist, is expected to induce a large increase in aldosterone, but not in cortisol levels in normal individuals (91). If a cortisol response to cisapride is found, a response to other 5-HT4R agonists such as zacopride or metoclopramide should be seen, but not to specific 5-HT-1,2,3 agonists. Although some specific 5-HT4R antagonists are currently under investigation, their availability is limited, but they should become very valuable in confirming the role of this abnormally expressed receptor.
C. Systematic clinical screening for ectopic/abnormal hormone
receptors
There has been only one report to date of the systematic clinical
screening of patients with adrenal CS for the presence of diverse
abnormal hormone receptors (240). In that study, 20 consecutive
patients with adrenal CS secondary to either bilateral macronodular
adrenal hyperplasia (n = 6), unilateral adenoma (n = 13), or
carcinoma (n = 1) were tested for evidence of an abnormal hormone
receptor. All six patients with AIMAH had a positive response to at
least one test, in addition to ACTH 124: two patients, to the mixed
meal (GIP-dependent); one patient, to GnRH (LH/hCGR) and cisapride
(5-HT4R); and three patients, to the upright
posture and vasopressin (1 ß-AR, 1 V1-AVPR, 1 ß-AR, and V1-AVPR).
In patients with unilateral adenoma, only one patient had a positive
response to upright posture, while three partial responses to either
mixed meals, vasopressin, or posture were also noted but were not
further characterized. In the patient with adrenocortical carcinoma or
in two patients with micronodular adrenal dysplasia (274), plasma
cortisol was not modified by any of the tests. Initial experience
suggests that the adrenal expression of various ectopic or abnormal
hormone receptors is frequently implicated in the pathophysiology of
bilateral macronodular adrenal hyperplasia (240), but less frequently
in unilateral adenoma (79). It must be noted that the initial protocol
used to date did not screen for many other G protein-coupled membrane
receptors, such as those for PTH, calcitonin, acetylcholine, dopamine,
opiates, prostaglandins, etc; it may thus become pertinent to
investigate these other potential abnormal receptors in the future.
| VII. Molecular Mechanisms of Ectopic/Abnormal Hormone Receptors |
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|
|
|---|
The ACTH MC-2 receptor gene, localized on human chromosome 18
(18p11.2), is highly expressed in the adrenal cortex and, at lower
levels, in fat tissue and skin (22, 279, 280). The proximal promoter
region (
1,000 bp) of the human ACTHR (hACTHR) gene is responsible
for the basal transcriptional rate and contains several potential
regulatory elements: one SP1 element, four AP1 elements, seven CRE
(cAMP-responsive element)-like regulatory elements, and three SF-1-like
elements (SF-35, SF-209, and SF-98) (23, 281, 282). Both SF-35 and
SF-98 sites were shown to be essential for the cAMP regulation of ACTHR
transcription. Although absolutely required, SF-1 is not sufficient for
ACTHR expression in the adrenals, since it is not expressed in gonads,
whereas both Leydig and ovarian cells express SF-1 (41). The well known
up-regulation of the receptor by its own ligand (36, 37, 283, 284, 285) is
probably mediated by one of the CREs. The same regulatory elements are
present in the proximal promoter of the mouse ACTHR, except for CREs,
which have been changed for GRE (GC-responsive element) sites (286). A
negative regulatory region (silencer), located between 1,236 and
908 from the transcription start site, prevents expression of the
receptor in heterologous systems or in non-SF-1-containing cell sites
(286). This suggests that other factors are needed for the receptor to
be expressed properly.
ß-Adrenergic receptors (ß-AR) are subject to extremely tight regulation. In addition to short-term regulatory phosphorylation of receptor proteins, their gene expression is also regulated. Cloning of the 5'-flanking region of human ß1-AR (chromosome 10q2426) revealed several potential thyroid response elements (TRE), GREs, and CREs (287). These putative response elements support the pathophysiological evidence that thyroid and GC hormones regulate ß1-AR by affecting receptor expression (287, 288). Hyper- and hypothyroidism have been associated with increases or decreases in ß1-AR number and activity. Thus, the presence of TRE in the 5'-flanking region of ß1-AR is consistent with these clinical conditions (289, 290). ß2-AR (chromosome 5q32) expression is up-regulated by GC in various tissues and is due to a direct increase in the rate of its transcription (291, 292). This is probably mediated by GRE, as demonstrated for hamster ß2-AR (293). In contrast, ß1-AR is down-regulated by GC. The stability of ß1-AR mRNA is not influenced by GC, but nuclear run-on assays have revealed that down-regulation is due to a decline in the relative transcription rate of the receptor (294). Homologous desensitization of ß-AR has been observed for the three receptor types, ß1-, ß2-, and ß3-AR (291, 295, 296). This is compatible with the presence of CRE in the promoters of both ß1- and ß2-AR (287). Moreover, ß-adrenergic stimulation causes not only down-regulation of ß-AR but also loss of coupling to Gs/AC effectors (297). In vivo investigations of GC effects on ß-agonist-induced down-regulation of ß1- and ß2-AR have shown that GC can prevent down-regulation of ß2-AR number and mRNA at the transcriptional level; the TF CREB may be involved (294).
LH/hCGR have also been reported in the human adrenal zona reticularis (249), although they are more highly expressed in gonadal tissues. The LH/CGR is one of the largest seven-transmembrane receptors (683 amino acids) as it harbors an unusually long extracellular ligand-binding domain (247). This receptor is encoded by two genes: gene I isolated from a lymphocyte library, and gene II isolated from a placental library (298). The four copies of hLHR genes are localized on chromosome 2p1621 loci. The two proximal 5'-untranslated regions have been well characterized (299, 300, 301) and differ by several base changes and a 6-bp deletion in the coding region (+55 to +60). The transcription initiation site is localized at position 176 bp for both promoter regions. Additional upstream transcription start sites have been identified in human testicular and choriocarcinoma JAR cells. These data suggest that tissue-selective LHR promoter utilization and gene (I or II) expression may underlie the specific pattern of LHR expression. TATA and CAAT-like boxes have been identified in human, but not in mouse and rat, promoters; the human promoter contains one CRE, seven AP1 sites, and one half-ERE site. Three negative control regions (NCRs), when complexed with proteins of JEG-3 cell nuclear extracts, disable the proximal promoter activity (300); these regions might be very important in nongonadal tissues.
The actions of vasopressin are mediated by three G protein-coupled membrane receptor subtypes. V2 receptors are expressed almost exclusively in renal collecting ducts to promote water permeability via activation of Gs and AC (302, 303). VIa (or VI) receptors are expressed in blood vessels, where they promote vasoconstriction (304), and in the liver, where they promote glycogenolysis (305), while VIb (or V3) receptors are located mainly in the anterior pituitary, but also in the adrenal medulla. VIa and Vlb receptors are coupled to various pertussis toxin-sensitive G proteins and activate PLA2, PLC, and PLD through activation of ligand-gated calcium channels (306). VIa receptors are also present in the adrenal cortex where they are involved in steroid secretion (see Section II). Dexamethasone increases the expression of VIa receptors in the rat liver and forebrain (307, 308). The elevation of mRNA levels precedes the rise in binding activity, suggesting a transcriptional effect. Isolation and analysis of the 5'-regulatory region of the rat VIa receptor have demonstrated that trans-acting factors such as CREB, AP-2, and GR are involved in the expression of the receptor gene (309, 310). At the protein level, GC have been shown to produce an early decrease in binding site density, followed later by an increase, which becomes more prevalent with time. Perhaps GC initially affects the stability of receptor protein or that of mRNA levels (307, 311). GC can also negatively regulate the stimulated expression of V1a receptor by a mechanism not involving GR-binding to DNA (310). Furthermore, it has been reported that GC amplify the vasopressin-induced transduction signal (increased IP accumulation in the presence of dexamethasone) (312, 313). This mechanism of regulation was demonstrated for the V1b receptor in the anterior pituitary where prolonged exposure to dexamethasone decreased the number of receptors, while increasing their coupling efficiency. Potentiation was found to be due, in part, to an increase in the guanylyl nucleotide-binding protein, Gq (314). The effect of GC on adrenal VIa receptors has not been studied.
The recent cloning of rat (214), hamster (215), and human GIPR (216, 217, 218) has revealed that it is a member of the secretin-VIP family of receptors. This gene is proposed to be involved in the pathogenesis of diabetes as GIPR knockout mice displayed glucose intolerance with impaired insulin secretion (218A ). The human GIPR gene is localized on the chromosome 19q13.3 locus and consists of 14 exons; it is expressed in several tissues, including the rat brain, fat, gut, vascular endothelium, and adrenals (214, 315, 316). In situ hybridization studies indicate that the GIPR is localized in the inner layers of the rat adrenal cortex (214); GIP is also able to stimulate AC and corticosterone synthesis in the rat adrenal cortex (317). In humans, the tissue distribution of GIPR mRNA has not yet been examined extensively, but has been discovered in the pancreas and brain, but not in spleen (210). Several splice variants of the receptor have been described in the human pancreas, of which one with a 27-amino acid insertion in the cytoplasmic tail is functional (216, 219). The rat GIPR has been shown to be desensitized by its own ligand in vivo and in vitro (318). The rat 5'-flanking region of the receptor gene has recently been cloned (319); it is a TATA-less promoter harboring one CRE, an octamer-binding site, three Sp1 sites, and an initiator element (319). Distal negative control sequences, not yet clearly identified, seem to confer cell-specific regulation of GIPR expression (319). The human GIPR promoter, however, has not yet been characterized.
5-HT4R-mediated stimulation of corticosteroid secretion is the only known endocrine effect mediated by this receptor. Activation of 5-HT4R augments AC activity and elevates cAMP. Homologous desensitization has been postulated to occur via a specific receptor kinase. Splice variants of 5-HT4R have been detected in several human tissues, and their tissue distribution suggests some degree of tissue specificity (320). These splice variants differ in their capacity to trigger the signal transduction cascade after receptor activation.
B. Potential mechanisms of ectopic or abnormal hormone receptors
The molecular mechanisms responsible for the ectopic or abnormal
expression and function of membrane receptors in adrenal CS have not
yet been identified. In fact, the important question of regulation of
the tissue-specific expression of genes is raised by this new
pathophysiology of adrenal CS. Several hypothesis can be proposed,
however (Fig. 5
). A gene rearrangement
could potentially lead to adrenocortical-specific, inappropriate
expression of a hormone receptor gene. Examples of this mechanism in
endocrine tumors include rearrangements described in subsets of
parathyroid adenomas (321), in GC-remediable aldosteronism (322), and
in papillary carcinoma of the thyroid (323). The PTH promoter has been
found to be recombined with the cyclin D gene, giving rise to the
prad-1 oncogene (321). The aldosterone synthase gene has been shown to
be fused with the 11ß-hydroxylase promoter, resulting in ectopic
production of aldosterone in zona fasciculata (322). In 25% of human
papillary carcinomas (up to 62% after exposure to Chernobyl
irradiation), a chromosomal break fuses the intracellular tyrosine
kinase domain of the growth factors receptor RET to one of at least
eight new promoters including H4, ELE1, R1
, NTRK1, RFG, and other
genes (324, 325); this results in constitutive dimerization and
activation of the tyrosine kinase of RET, bypassing the requirement for
ligand binding. None of the ectopic hormone receptors identified to
date in adrenal CS is located on the same chromosome as the ACTHR
promoter; gross gene rearrangements have not been reported to date.
More discrete mutations in the promoter regions of the membrane hormone
receptor could also greatly increase the expression of a receptor
normally expressed at such a low level that it would not play a
significant role in steroidogenesis. A point mutation in the promoter
region of the hormone receptor could generate an appropriate binding
site for an adrenocortical-specific TF/co-activator complex, leading to
ectopic expression (Fig. 5A
).
|
Another interesting hypothesis emerges from a recent study by Kero et al. (327), who observed that transgenic mice expressing bLHß-CTP (a chimeric protein of ß -subunit fragments of bovine LH and hCG) in their pituitary develop adrenal CS in addition to polycystic ovaries and ovarian tumors after chronically elevated serum LH levels. It was shown that this resulted from ectopic expression of LH/CG receptors in the adrenal cortex, not detectable or functional in control mice. Since this induction is abolished by gonadectomy, it was proposed that elevated estrogens and PRL levels were responsible for inducing the illicit expression of the LH/CG receptor in the adrenal cortex. This observation would thus raise the possibility that the "ectopic expression" of a receptor may not require a mutation of cis- or trans-acting regulators, but may result from exaggerated stimulation of a gene that is normally silent.
The presence of abnormal membrane hormone receptors in unilateral
cortisol-secreting adenomas may arise from the monoclonal expansion of
a primary adrenocortical cell that acquired a somatic mutation, leading
to the abnormal expression and function of that receptor at the
postzygotic stage of adrenal cortex development (Fig. 2
); most studies
confirm the monoclonal composition of human adrenocortical tumors (157, 163). In patients with ectopic membrane hormone receptors in bilateral
macronodular adrenal hyperplasia, the mutational event must have
occurred very early during embryogenesis so that every cortical cell of
both adrenals would be affected by the defect, which is polyclonal.
There have been rare reports of familial AIMAH (179, 180, 181, 182), and in only
one case, an abnormal response to LVP was demonstrated in one sibling
(182); thus, abnormalities of receptor expression in these syndromes
may frequently be the consequence of somatic mutations but, in some
cases, could also be germline mutations. In the case of a very early
mutational event resulting in AIMAH, the abnormal expression could
affect diverse tissues so that polymorph aberrant manifestations would
be expected. This was the case in the patient with
vasopressin-dependent CS (80) who also displayed an abnormal vascular
response to AVP and decreased hypothalamic AVP release during postural
hypotension. One patient with GIP-dependent CS and AIMAH suffered from
psychiatric dysfunctions that persisted even after correction of the
hypercortisolism (200); since GIPR have been shown to be expressed in
the brain (214), it is possible that the brain GIPR is also altered.
The McCune-Albright syndrome is an example of a somatic mutation
occurring during embryogenesis and leading to defects in the adrenal
cortex as well as in several other tissues (172); it is still unclear
how a somatic mutation could affect all cells in a polyclonal mode in
one case (ectopic hormone receptors in AIMAH and CS) and result in a
mosaic or oligoclonal pattern of distribution in another case
(McCune-Albright syndrome).
The majority of ectopic or abnormal hormone receptors in adrenocortical
tumors or hyperplasias (8, 9, 10) belong to the G protein-coupled receptor
superfamily. Studies of the second messengers implicated in
ectopic/abnormal hormone receptors in adrenal CS suggest that they
regulate steroidogenesis by mimicking the cellular events triggered
normally by ACTHR activation (Fig. 1
). It is thus expected that only
ectopic hormone receptors capable of coupling efficiently to the
intracellular signaling systems present in adrenocortical cells
(i.e., those for ACTH, V1-AVP, etc.) will be able to
regulate steroidogenesis aberrantly. Certain receptors may be involved
more frequently than others, however, if they share more
characteristics of the promoters or TFs essential for adrenal cell
type-specific tissue expression. It is thus noteworthy that GIPR and
the ß-AR are expressed normally and are functional in the adrenal
fasciculata cells of rodents; it will be interesting to compare the
structures of promoters and TFs between species. The LH/hCGR is
expressed in the human adrenocortical reticularis during embryonic
life; it remains to be seen which events render its expression possible
in the fasciculata in adrenal CS (251). Similarly, the
5-HT4R is usually very efficiently coupled to
aldosterone synthesis in the human glomerulosa and already possesses
some tropism for fasciculata cells; increased functional coupling to
cortisol secretion may require only the inactivation of a relative
silencer in fasciculata cells.
C. Role of ectopic hormone receptors in adrenocortical cell
proliferation
What is the role of abnormal hormone receptors in altered cell
growth and tumorigenesis? One could postulate that the primary event is
a mutation resulting in aberrant adrenal expression of the receptor,
leading to increased proliferation and eventually to increased hormone
production. Alternatively, it can be proposed that the primary event is
an unknown proliferative one resulting in cell dedifferentiation with
resultant expression of "embryonal" type genes, including one or
several hormone receptors. In either hypothesis, it is clear that a
relatively long time period is necessary before phenotypic expression
of the abnormal hormone receptor becomes evident. This is particularly
true for AIMAH, as several decades are necessary before the hyperplasia
and hyperfunction become clinically manifest. This may be secondary to
the transient occupation of the receptor by the ligand, as illustrated
by the cases of GIP (Fig. 2
) and LH-dependent (Fig. 3
) CS. In
GIP-dependent CS, the adrenal tissues are stimulated only briefly but
repeatedly after each food ingestion; in LH/hCG-dependent CS, the
hyperplasia and hyperfunction occurred only after intense and prolonged
exposure to the ligands, i.e., during pregnancy for hCG, or
after menopause for LH. Reversal of the hyperplasia between pregnancies
would favor the hypothesis that the ectopic receptor is a primary event
rather than one that is secondary to another proliferative event;
however, this awaits clear demonstration of adenoma or AIMAH regression
after complete blockade of the ectopic receptor. There is also
indication, based on cases of preclinical cortisol production in
bilateral macronodular disease, that steroidogenesis can be relatively
inefficient, despite significant proliferation. This suggests poor
steroidogenic enzyme activities in the adrenal lesions or that the low
expression of abnormal receptors is better coupled to proliferative
signals than to hormone synthesis.
The elucidation of this question requires better understanding of the factors regulating normal adrenal gland development. Knowledge of the ontogeny of steroidogenic tissues (adrenals and gonads) was provided by the identification of tissue-specific TFs (328, 329). Indeed, by using SF-1 as a marker, it became possible to trace steroidogenic cells back to the earliest stage of differentiation (330). Investigation of the spatiotemporal expression of SF-1 revealed the existence of the adreno-genital primordium (AGP) which is composed of a SF-1-immunoreactive single cell population (for review see Ref. 331). This structure lies between the coelomic epithelia of the urogenital bridge and the dorsal aorta. The AGP then gives rise to adrenocortical and gonadal primordia, which both express SF-1. Studies in SF-1 knockout mice have shown that the earliest stages of urogenital ridge development occur normally; however, regression of the adrenals and gonads is observed as soon as gonadal sexual differentiation takes place (66). These results suggest a complex cascade of transcriptional events for establishment of the endocrine axis. The adrenocortical primordium gives rise to the adrenal cortex that differentiates into three zonae (glomerulosa, fasciculata, and reticularis). The adrenal medulla is composed of neural cells (SF-1-negative cells) that have migrated from a dorsal root ganglion to the adrenal primordium. In adult mice, SF-1 is expressed in adrenocortical, testicular Leydig, ovarian theca, and granulosa cells, and, at a lower level, in spleen and pituitary gonadotropes (reviewed in Ref. 41).
DAX-1 (dosage-sensitive sex reversal, AHC critical region on the X chromosome, gene 1) is another steroidogenic-specific TF involved in the adrenal cortex and gonads, as demonstrated by disorders due to DAX-1 mutations (332, 333, 334). DAX-1 belongs to the orphan nuclear receptor superfamily as does NGFI-B and SF-1 and it acts as a transcriptional repressor. However, the protein is atypical since it possesses no DNA-binding domain, suggesting possible interactions with other TFs. Indeed, DAX-1 has an expression profile similar to that of SF-1, suggesting a functional correlation between these two proteins (335, 336, 337). Moreover, SF-1 was shown to be a critical regulator of DAX-1 expression, as functional SF-1-binding sites have been identified in the promoter region of DAX-1 gene (338, 339). Another transcriptional repressor has been shown to play a role in adrenal development. Initially designated as an essential actor throughout nephrogenesis (340, 341), the Wilms tumor suppressor gene (WT1) has recently been implicated in adrenogenesis (342). Unlike SF-1, WT1 expression is not detectable during adrenal cortex formation (343, 344) but is in the developing kidney and urogenital system. Taken together, these results suggest, first, that the WT1 gene may be expressed in a very transitory manner in adrenocortical precursor cells, and second, that WT1 activity may be required at early steps of adrenal development, probably in the AGP stage. Functional interactions between SF-1, DAX-1, and WT1 have been demonstrated for transcriptional regulation of the Mullerian inhibiting substance (MIS) sex-specific gene in vitro (344). Such combinational regulation may occur for the expression of genes determining the fate of the AGP. It should be interesting to determine whether any alterations in SF-1, DAX-1, or WT1 could be present, particularly in cases of AIMAH with ectopic membrane hormone receptors.
The concept of abnormal G protein-coupled receptors and/or postreceptor
events leading to increased cAMP and proliferation is now well
established (Table 4
, reviewed in Refs.
345, 346, 347), especially in somatotroph and thyroid cells (348, 349).
Stimulation of G-protein-coupled receptors, alone or in association
with tyrosine kinase receptors, is known to evoke powerful mitogenic
signals via G protein-mediated activation of ras (346). Thus, altered
activity at any step of the transduction signal cascade may predispose
to tumor formation. Transgenic mice with thyroid-specific expression of
adenosine A2 receptor (which activates AC via Gs
protein) develop thyroid hyperplasia and severe hyperthyroidism (350),
clearly demonstrating that in vivo constitutive activation
of the cAMP cascade in thyroid cells is sufficient to stimulate
autonomous hyperfunction and uncontrolled cell proliferation. There are
many examples of hormone receptor mutations involved in endocrine
pathologies (Table 4
). Some include somatic or germline constitutive
mutational activation of the TSH receptor, resulting in
hyperfunctionning thyroid adenomas and hyperplasias (351, 352);
familial male precocious puberty (characterized by Leydig cell
hyperplasia and testosterone production) is due to constitutive
activation of LH/hCGR (353). At the G protein level, the
mosaic-activating mutation of Gs
leads to
McCune-Albright syndrome (172); activating mutations of inhibitory
G
i protein (Gip) have
been identified in some, but not all, adrenocortical and ovarian tumors
(355), and Gs
overexpression has been shown in
insulinomas and other endocrine tumors (356). There are examples of
transgenic mice with cardiac overexpression of
ß2-AR or Gs
that
display enhanced cardiac function and develop myocardial fibrosis
(357). However, it must be stressed that cAMP is not mitogenic in all
cell types. Counterregulatory mechanisms are initiated in response to
persistently elevated cAMP levels. This was the case for transgenic
mice expressing gsp in pancreatic ß-cells (358) where inhibitors of
phosphodiesterases were required to obtain high cAMP levels and
enhanced insulin secretion. In the Y1 mouse adrenocortical cell line
transfected with ß2-AR, ectopic receptors have
been found to be efficiently coupled to steroidogenesis, but cell
growth has not been studied (359).
|
-subunit promoter/simian virus 40
T-antigen fusion gene (360); it remains to be seen whether the
expression of ectopic adrenocortical receptors, in the absence of other
oncogenic events, is sufficient for adrenal overgrowth. Future animal
models such as transgenic mice expressing ectopic membrane hormone
receptors in the adrenal cortex will be informative in this regard.
This is already supported by the demonstration of bilateral adrenal
hyperplasia and CS in the mice transgenic for bLHß-CTP with ectopic
adrenal expression of LH/CGR (327). What is the cell of origin in which the receptor is expressed abnormally? Based on the profile of steroids produced, it appears that it can occur in well differentiated cells of the fasciculata/reticularis (pure cortisol- or mixed cortisol-/androgen-secreting adenoma), and in cells from the reticularis (pure androgen-secreting adenoma); the three classes of adrenal steroids are sometimes secreted in macronodular hyperplasia, suggesting that all zonae are affected. It remains to be seen whether some cases of unilateral adenomas or bilateral hyperplasia in primary hyperaldosteronism can also be secondary to ectopic hormone receptors.
No constitutive activating mutations of the ACTHR have yet been found in adrenocortical neoplasms or hyperplasias (361). Recent studies suggested that ACTHR could act as a tumor suppressor gene in adrenal tumorigenesis (362) in a way similar to p53, which is involved in many tumor types, including adrenocortical tumors (363). Loss of heterozygosity of the ACTHR gene was shown to be associated with high malignancy or the absence of secretion in a subset of human adrenocortical tumors. Furthermore, lower expression of ACTHR was found in adrenocortical carcinomas compared with adrenocortical adenomas from patients with CS (364, 365). ACTH is known to be a differentiating factor with low potential for promotion of cell proliferation, as demonstrated by in vitro experiments. It has thus been speculated that a defect in the ACTHR signal cascade could result in dedifferentiation and increased cell proliferation (362). Obviously, much work remains to be done to better understand the mechanisms underlying tumorigenesis of the adrenal cortex.
| VIII. Ectopic/Abnormal Hormone Membrane Receptors in Nonadrenocortical Tumors |
|---|
|
|
|---|
Matsukura et al. (370) found aberrant AC stimulation in four GH-secreting pituitary adenomas by TRH (two of four), GnRH (two of four), norepinephrine (three of four), dopamine (one of four), glucagon (one of three), or PGE1 (four of four); in one ACTH-secreting pituitary adenoma, AC was stimulated by GnRH, norepinephrine, and glucagon, but not by TRH. The paradoxical stimulation of GH or ACTH after the GnRH or TRH tests in vivo in patients before surgery correlated well with the AC stimulation in vitro. The AC of two ectopic ACTH secreting tumors (gastric carcinoid and malignant thymoma) was also stimulated by TRH, GnRH, norepinephrine, epinephrine, serotonin, and PGE1 (370).
The frequently observed paradoxical increase in GH in acromegalic patients after administration of TRH, or in a lesser proportion, of GnRH (371, 372, 373) and the AC stimulation found in vitro (370) suggested the presence of ectopic TRH or GnRH receptors in GH-secreting pituitary tumors. The expression of TRH receptors type 1 has been confirmed in GH-secreting adenomas (374), where the structure of the receptor does not appear to be mutated (375); the TRHR-1 is normally expressed in rat somatotroph cells (376), and it is unknown whether the abnormal response of GH in acromegaly results from ectopic expression of one of the TRH receptors, or rather from abnormal coupling of this receptor to GH secretion in adenoma cells. In a preliminary report, the paradoxical increase in GH following oral glucose in acromegaly was found to result from aberrant GH-tumor response to GIP (376A ); this would suggest that ectopic GIPR could also occur in acromegaly. Epidermal growth factor (EGF) receptor is overexpressed in several types of human cancers including aggressive GH-secreting tumors (377).
As a corollary to the ectopic expression of LH/hCGR in the adrenal cortex, the stimulation of androgen secretion in patients with ovarian arrhenoblastomas, after administration of ACTH, and their suppression by dexamethasone indicate the ectopic expression of ACTHR in some of those tumors (378, 379).
In a sporadic human medullary thyroid carcinoma (MTC), Matsakura
et al. (380) found that the AC was activated by TRH,
glucagon, epinephrine, norepinephrine, and serotonin, but not by TSH,
ACTH, or PRL. A large number of studies have now evaluated the
expression and function of hormone and growth factor receptors in MTC
(381, 382). It is problematic to distinguish which of the receptors
identified are indeed ectopic, as frequently, the search for their
expression in normal C cells has not been performed. Mutations of the
normally C cell-expressed RET protooncogene (eutopic receptor) are
present in almost all cases of genetic forms of familial MTC and MEN-2
(multiple endocrine neoplasia, type 2), and in a proportion of
sporadic MTC cases (somatic), and play a crucial role in initiation of
C cell proliferation (323). Clearly, other receptors contribute to the
development and progression of MTC, e.g., the trk family,
neurotrophin receptors, where the type trkB is reduced, while trkC
expression is increased during the progression of the disease (381).
Some of these proliferative-related receptors are expressed also in
normal thyroid; this appears to be the case for transforming growth
factor-
(TGF-
and EGF), as well as for their common EGF receptor
(382). However, EGF binding protein, particularly EGFBP-2 and -3, are
detected only in MTC (382). Rat MTC cell line 6/23 also expresses
GLP-1 receptor, VIP receptor, and PACAP receptor (383); in
addition, several splice variants of PACAP were expressed in 6/23 cell
line. The GLP-1 receptor expression is responsible for glucagon effect
on calcitonin secretion via cAMP stimulation (384). Additional
receptors in which ectopic or increased expression may be related
to the progression of the disease include progesterone
receptors, which are focally detected in all studied cases of MTC
without the concurrent presence of estrogen receptors (385). Expression
of gastrointestinal hormones and their receptors, particularly those of
CCK-B/gastrin, also received attention in MTC. Thus, CCK-B/gastrin
receptors were detected in all biopsy specimens, while they were not
found in normal thyroid tissues or in other thyroid tumors such as
follicular adenoma, papillary carcinoma, or anaplastic carcinoma (386).
Therefore, the presence of CCK-B/gastrin receptor in MTC may have
clinical implications. Much attention has been paid, over the last
decade, to somatostatin receptor expression in MTC and many other tumor
types. The genomic structure and transcription regulation of the
various types of somatostatin receptors are now better understood in
MTC (387).
Many other receptors have been described, during the last decade, as
being expressed in the adrenal medulla tumors without apparent clinical
evidence of their ectopic activities. Such is the case for the ANP
receptor and its effect on catecholamine release in human
pheochromocytoma (388). Most of the receptors studied more recently
have, at least, a potential relevance for control of proliferation.
Thus, IGF-II itself is produced and released by the adrenal and is
accompanied by the presence of IGF-II R in pheochromocytomas (389). As
the ectopic expression of Src homology 2 (SH2) and SH3-containing
oncogenic adaptor protein v-Crk in PC12 cells results in EGF-inducible
neuronal differentiation, v-Crk was studied and demonstrated able to
regulate the strength of a tyrosine kinase signal that leads to
prolonged activation of Ras and MAP kinase, respectively (390).
Pheochromocytoma shares the expression of several genes with MTC; one
example is TGF
gene and its receptor EGFR (391). Both of these
tumors express these receptors in vivo and in
vitro, and it has been suggested that TGF
is involved in the
regulation of tumor cell growth. Since the signaling pathway from the
TrkA receptor via the MAP kinase is not altered in PC12 cells, it has
been proposed that p300 could play a pivotal role in triggering the
antimitogenic effect of NGF and neuronal differentiation (392).
Since all cells are regulated in their function and proliferation by a series of hormone and growth factors that signal the cells via membrane receptors, it appears quite plausible that several other examples of ectopic or abnormal membrane receptors will be identified in various hyperplasias and tumors in diverse endocrine and nonendocrine human tissues.
| IX. An Opportunity for New Pharmacological Therapeutic Strategies |
|---|
|
|
|---|
Pharmacological blockade of postprandial GIP release with octreotide
was attempted in a few patients with GIP-dependent CS as an alternative
to surgery (Table 2
; Refs. 201, 208, 209). During the first months
of subcutaneous octreotide administration before each meal, clinical
and biological improvements were documented, but long-term treatment
proved to be ineffective. It is presumed that the escape of octreotide
efficacy was secondary to down-regulation of somatostatin receptors in
GIP-secreting intestinal cells. Thus, adrenalectomy remains the
long-term treatment of choice for this syndrome until specific GIPR
antagonists become available. Short-term use of the oral V1-AVPR
antagonist OPC-21268 for 8 days decreased urinary free cortisol levels
in a patient with vasopressin-responsive AIMAH and CS (239).
In the patient with catecholamine-dependent CS and bilateral AIMAH (86), initial treatment with propranolol up to 320 mg daily was able to considerably reduce cortisol secretion; however, urinary cortisol levels remained approximately twice the upper limit of normal, and it was decided to remove one of the two very large adrenals surgically. It then became possible, upon restoration of propranolol administration, to completely normalize cortisol production. Interestingly, the control of hypercortisolism was followed by a decreased requirement in the dosage of the ß-blocker from 320 mg to 20 mg of propranolol daily, as higher doses were causing adrenal insufficiency. GC are known to stimulate ß2-AR transcription (292) via GRE located in promoters of the target genes (293). The normalization of cortisol levels may have decreased ß-AR density, which would explain the lower requirement for the antagonist. Propranolol therapy did not reduce the size of the remaining adrenal even after 3 yr of follow-up; however, the minimal dose of propranolol necessary to maintain normal cortisol production was administered, without blocking the receptors completely. This constituted the first example of long-term pharmacological blockade of an ectopic adrenal membrane hormone receptor.
In the patient with LH/hCG-dependent AIMAH and CS, the suppression of
endogenous LH levels with chronic, long-acting leuprolide acetate
controlled the hypercortisolism (Fig. 3
) and avoided bilateral
adrenalectomy (251). Leuprolide acetate, a long-acting GnRH agonist,
initially stimulated gonadotropin release, which increased cortisol
production for 1 week; this was followed by suppression of endogenous
LH levels and normalization of cortisol production. Despite complete
suppression of endogenous LH levels, the patient did not present
cortisol insufficiency. It is possible that basal cortisol production
was maintained by serotonin stimulation, since there was also evidence
of abnormal 5-HT4R function in the same adrenals.
The absence of regression of bilateral adrenal hyperplasia, despite
chronic suppression of endogenous LH, indicates that its size was
maintained by abnormal function of 5-HT4R, or
that aberrant receptors regulate steroidogenesis but not cell
proliferation. It will be interesting to study the effects of a
specific 5-HT4R antagonist in this patient when
it becomes available. A GnRH analog has previously been used
successfully in long-term suppression of testosterone-secreting ovarian
tumor (393).
Further studies will probably identify a larger diversity of hormone
receptor abnormalities and should eventually allow the use of new
pharmacological tools to inhibit either the production of endogenous
ligands or block the receptors with appropriate specific antagonists
(Table 5
). Since it is also possible to
detect the presence of ectopic/abnormal hormone receptors at the stage
of preclinical steroid hormone production (242), it will be of great
interest to investigate whether the progression of adrenal tumors or
hyperplasias can be prevented by these new pharmacological approaches.
|
| X. Summary and Conclusions |
|---|
|
|
|---|
| Acknowledgments |
|---|
| Footnotes |
|---|
1 Supported by a grant from the Medical Research Council of Canada
(MA-10339). ![]()
| References |
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