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Molecular and cellular regulation of primary macronodular adrenal hyperplasia
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Molecular and cellular regulation of primary macronodular adrenal hyperplasia Maria Candida Barisson Villares Fragoso1 and Claudimara Ferini Pacicco Lotfi2 Abstract
Available online 21 August 2019
Primary macronodular adrenal hyperplasia (PMAH) is an adrenal cause of Cushing’s syndrome, the clinical and the pathological features of which were described for the first time by Marvin Kirschner et al. in 1964 [1]. At that time, the authors suggested the following as an explanation for this heterogeneous adrenal disorder: a more tenable hypothesis would be that multinodular hyperplasia with macronodules represents an abnormal outcome of adrenal stimulation, with eventual concomitant, possibly localized, loss of adrenal secretory efficiency. Over the 55 years since PMAH’s identification, our knowledge has advanced to clarify the mechanisms involved in the pathophysiology of this disorder. Up to now, diverse molecular events have been proposed to explain the enhanced cortisol secretion, cell proliferation, and development of macronodules that occur in PMAH. Nevertheless, the precise sequence of events and molecular mechanisms underlying in this condition remaining incompletely elucidated. Recent studies indicate that PMAH is more genetically determined than previously thought according to the bilateral nature of the disease. In addition, the description of familial forms, with an autosomal dominant pattern suggested an inherited genetic etiology for PMAH. The purpose of this review is to summarize the primary insights concerning the molecular and cellular regulatory mechanisms of PMAH.
https://doi.org/10.1016/j.coemr.2019.08.007
Addresses 1 Unidade de Suprarrenal, Disciplina de Endocrinologia e Metabologia, Hospital Das Clinicas da Faculdade de Medicina da USP, Instituto Do Câncer Do Estado de São Paulo – ICESP, University of Sao Paulo, Brazil 2 Instituto de Ciências Biomédicas Do Departamento de Anatomia, University of Sao Paulo, Brazil Corresponding author: Barisson Villares Fragoso, Maria Candida ([email protected])
Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121 This review comes from a themed issue on Adrenal Cortex Edited by André Lacroix and Enzo Lalli For a complete overview see the Issue and the Editorial
Funding statement from Fundac¸˜ao de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP) to MCBVF # 2015/50192-9. Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121
2451-9650/© 2019 Elsevier Ltd. All rights reserved.
Keywords Adrenal hyperplasia, PMAH, Cushing’s syndrome, Pathophysiology.
Introduction Primary macronodular adrenal hyperplasia (PMAH) was first described by Marvin Kirschner et al. in 1964 [1] and it is considered a rare cause of endogenous hypercortisolism occurring in less than 2% of all cases of Cushing’s syndrome. Nevertheless, it has been more frequently diagnosed than in the past because of increasing of radiological abdominal exams showing adrenal incidentaloma and because of the recognition of genetic inheritance in more than 50% of patients. Few years ago, inactivating germline and somatic mutations in the armadillo repeat-containing 5 (ARMC5) gene was accepted as a major genetic cause of patients with PMAH.
Cellular characteristics PMAH Macroscopic examination of the macronodular adrenal hyperplasia shows characteristic increase in adrenal mass, which can reach up to 100 times the normal weight and small to large nodules with yellow coloration [2]. Microscopically, this presents as nonpigmented nodules composed of two groups of cell types with no internodular atrophy [3]. Spongiocytes group of cells with clear lipid-rich cytoplasmic regions are a predominant component of the nodules (personal information), with compact cells forming an island-like structure with lipid-poor cytoplasmic regions. No mitosis or nuclear variability with respect to size and shape is observed in either cell types. Immunolocalization of steroidogenic enzymes in both cell types showed 3b-hydroxysteroid dehydrogenase/D5 4 isomerase distribution within spongiocytes, whereas the steroid 17 alpha-hydroxylase/ 17e20 lyase (P450c17) is detected in compact cells [4]. The inefficient steroidogenesis in this celleenzymatic profile could in part explain the mild hypercortisolism observed in PMAH patients despite enlargement of the adrenal glands [5]. www.sciencedirect.com
Molecular and cellular regulation of PMAH Fragoso and Lotfi
Aberrant G-protein-coupled membrane receptors Under physiological condition, adrenocorticotropic hormone (ACTH) is the primary regulator of the steroidogenesis. ACTH acts through of its melanocortin 2 receptor (MC2R), a G protein-coupled receptor (GPCR). This complex hormone-receptor activation determines cAMP/protein kinase A (PKA) pathway stimulation and increases steroidogenesis and cell proliferation in the adrenal cortex [5]. The presence of aberrant ectopic or eutopic hormone receptors excessively expressed in the adrenal cells in PMAH, despite suppressed ACTH levels, present the capacity to stimulate steroidogenesis and has been recognized as part of the pathophysiology of PMAH [6]. Several GPCRs have been described in PMAH nodules such as the receptor for luteinizing hormone (LH)/ human chorionic gonadotropin (hCG), the serotonin receptor (5-HT4 and 5-HT7 receptors), the leptin receptor, glucose-dependent insulinotropic peptide (GIP), catecholamine (b-adrenergic receptor), vasopressin (V1eV2eV3e vasopressin receptor), glucagon, and angiotensin II. However, whether high GPCR expression and their response to specific ligands is a primary event or secondary phenomenon resulting from cell proliferation and dedifferentiation is not entirely clear [6,7].
GIP and glucagon effects in PMAH cells Hamet et al. in 1987 proposed a humoral factor induced by eating as responsible for periodic adrenal cortisol secretion [8] who observed that plasma fasting cortisol was low in the morning and increase following meal ingestion. This finding subsequently confirmed by Lacroix et al. in 1992 [9]. In vivo physiological concentrations of GIP stimulate cortisol production in PMAH patients in whom the presence of GIP receptors was suggested by adrenal imaging after injection of [123I]GIP. In dispersed cells from PMAH patients, GIP mediated cortisol secretion in contrast with normal adult or fetal adrenal cells. In addition, adrenal tissues from patients with GIP-dependent Cushing’s syndrome present overexpressed nonmutated GIP receptors. In normal human adrenal glands, glucose-dependent insulinotropic polypeptide receptor (GIPR) is barely expressed and is not related to efficient steroidogenesis [10]. However, GIP stimulates thymidine incorporation in PMAH adrenal cells but not normal cells [11]. In addition, activation of the GIPR receptor in PMAH patients with GIP-dependent adrenal Cushing’s syndrome is functionally coupled to cAMP signaling and triggers adrenal cell proliferation [6]. More recently, the molecular pathogenesis of GIP-dependent Cushing’s syndrome was shown to occur through monoallelic transcriptional activation of GIPR driven by chromosome 19q13 rearrangements [12].
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Together with GIP and others structurally related peptide hormones (glucagon-like peptides, vasoactive intestinal peptide, pituitary adenylyl cyclase-activating polypeptide, and growth hormone-releasing hormone) glucagon activates receptors coupled to the cAMP/PKA pathway [13]. The early observation that adenylyl cyclase activity is enhanced by glucagon in PMAH [14] led to hypothesis that illegitimate glucagon receptors are associated with Cushing’s syndrome. This finding was supported by an abnormal plasma cortisol response to glucagon in 58% of patients with PMAH as well as subclinical hypercortisolism [15]. More recently, evaluation of glucagon’s role in the regulation of cortisol secretion in PMAH patients has revealed the presence of glucagon receptor-like immunoreactivity in clusters of spongiocytes in adrenal tissues from patients who were sensitive in vivo to glucagon [16].
LH and LHCGR effects In 1999, Lacroix et al. [17]described a patient with Cushing’s syndrome because of PMAH with cortisol production stimulated in vivo by LH and hCG during her pregnancies, which became constant only after menopause. This suggests a functional LHCG receptor coupled to steroidogenesis. This receptor activates adenylyl cyclase and phospholipase C to stimulate gonadal steroidogenesis and the zona reticularis in the human adrenal cortex [18], whereas hCG stimulates the secretion of dehydroepiandrosterone sulphate in human fetal adrenal cells [19]. In this case, suppression of endogenous levels of LH with leuprolide acetate was able to normalize cortisol production. In mice, gonadectomy induces hyperplasia and tumorigenesis, apparently triggered by elevated postgonadectomy levels of LH/hCG, followed by ectopic upregulation of Luteinizing hormone/choriogonadotropin receptor (LHCGR). However, gonadectomy in tumorigenic, DBA/2J, and nontumorigenic C57BL/6J mice induced similar upregulation of adrenal LHCGR in both indicating that ectopic expression of this receptor is not the direct cause of tumors [20]. Similarly, analysis of aberrant LHCGR expression in different human adrenal hyperplasia patients (7 with PMAH, 5 with primary pigmented nodular adrenocortical disease and 8 with diffuse adrenal hyperplasia secondary to Cushing’s disease ) showed no differences between LHCGR expression among all tissues examined [21]. Interesting, there is a description of two cases with PMAH whose adrenal lesions expressed LHCGR and who had response to leuprolide acetate treatment even in the absence of increased cortisol. In addition, a study described novel features of gene expression LHCGR-mediated, pregnancy-induced through mechanism mutation-independent by hCG activated. In this study, a young prim-gravid patient with over Cushing’s syndrome within a week after delivery, the signs and symptoms of Cushing’s syndrome regressed, and they recovered during a new pregnancy [22]. Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121
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Taken together whole, the data above indicate that circulating hormone levels may influence cortisol release and contribute to the hypercortisolism observed in PMAH.
Activation of the cAMP/PKA pathway Activation of the cAMP-PKA pathway is the central mechanisms associated with PMAH. ACTH acts as a regulator of steroidogenesis and adrenal proliferation through the melanocortin 2 receptor, MC2R [23]. Activating mutations in the conserved MC2R gene lead to constitutive activation of the cAMP-PKA pathway, which controls the autonomy of adrenal gland; however, this mechanism is considered an extremely rare cause of PMAH [23]. Heterotrimeric G protein in the active state can modulate the activity of the ligands and their 7M receptors, increasing cAMP-PKA pathway activation. Somatic mutations of the gene encoding the alpha subunit of stimulatory G protein, GNAS, result in constitutive activation of adenylate cyclase in specific codons. These so-called gsp somatic mutations were previously described in patients with McCune-Albright syndrome associated with Cushing’s syndrome [24]. Interesting, these gsp mutations have also been described in adrenal nodules from PMAH patients, and while considered, a rare molecular event is sufficient to induce adrenal hyperplasia and cortisol secretion [25,26]. Phosphodiesterase’s (PDE) enzymes families are considered key regulators of intracellular cAMP levels. Recent studies have shown that the presence of inactivating germline mutations and allelic variants in PDE11A may increase susceptibility to PMAH [27]. In HEK293 cell lines, these allelic variants exhibited higher cAMP levels than wild-type cell lines, suggesting that these mutations cause reduced cAMP hydrolytic activity. Somatic activating mutations in the PRKACA gene, which encodes the catalytic subunit alpha PKA, were identified in 40% of adrenal adenomas producing cortisol. Interesting, germline duplications on a genomic region of chromosome 19 that included PRKACA were identified in PMAH patients [28]. Further studies in large cohorts of patients are needed to determine the frequency and effect of these molecular events on the pathogenesis of PMAH.
Paracrine/autocrine regulation of cortisol production in PMAH More recently, the notion has emerged that, in addition to membrane G-protein-coupled receptors, there are Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121
abnormal paracrine/autocrine factors in PMAH tissue distinct from the physiological paracrine/autocrine systems occurring in the normal adrenal gland. These abnormal factors lead to abnormal intraadrenal stimulatory loops and cortisol hypersecretion [29]. From studying, autocrine/paracrine/regulation of cortisol secretion in PMAH, a new concept of paracrinopathy emerged that was proposed in 2015 by Lefebvre et al. [30]. Some of the important autocrine/paracrine regulatory signals of cortisol secretion in PMAH are discussed in more detail below.
Serotonin In the normal human adrenal gland, 5hydroxytryptamine (5-HT) is produced and released by mast cells located in the subcapsular adrenal region, which stimulates corticosteroids secretion through the activation of 5-HT4 receptor positively coupled to adenylyl cyclase and calcium influx [31,32]. In situ hybridization in the normal adrenal cortex revealed that 5HT4 receptor mRNA was expressed in the zona glomerulosa, and a weaker hybridization signal was also visualized in zona fasciculata/reticularis cells [33,34]. In vitro studies demonstrated that 5-HT is efficient at stimulating aldosterone but is less efficient at stimulating cortisol [35]. In agreement with in vitro studies, administration of 5-HT4 receptor agonists in healthy patient induced increased aldosterone levels without affecting cortisol levels [35]. Distinct from normal adrenal glands, where the source of 5-HT is mast cells, immunohistochemistry analysis in PMAH tissues revealed abnormal synthesis of 5-HT in clusters of steroidogenic cells, suggesting an autocrine/ paracrine mechanism for cortisol production by 5-HT [36]. Clinical studies and analysis in PMAH tissues reported over expression of the 5-HT4 receptor, indicating mediation of the 5-HT4 receptor by serotonin’s action [37,38]. However, in some PMAH tissues, eutopic 5HTR4 expression was found in addition to ectopic expression of 5HTR7 receptor in the plasma membrane of steroidogenic cells. Both receptors are positively coupled with adenylyl cyclase, mediating corticosteroid-induced 5HT in PMAH through the cAMP/PKA pathway [39]. More recently, a study showed that in PMAH, high expression of TPH1 (5-HT synthesizing enzyme tryptophan hydroxylase) and 5HT4R was associated with ACTH-positive cells suggesting ACTH as a regulator of these proteins via an autocrine/paracrine mechanism. In contrast, 5-HT7R seems less dependent on the local synthesis of ACTH [40]. In summary, distinct from the normal adrenal gland, the presence of aberrant 5-HT production and illicit www.sciencedirect.com
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expression of serotonin receptors improve the 5-HT autocrine/paracrine pathway and hypercortisolism in PMAH.
Arginine vasopressin The arginine vasopressin (AVP) released by hypothalamic neurons stimulates ACTH production and glucocorticoid production in the adrenal glands. However, AVP is also released by chromaffin cells from adrenal medulla acting as a paracrine regulator of glucocorticoid production [41]. Vasopressin type 1a (V1a) receptors are present in the adrenal cortex and act via heterotrimeric G proteins phospholipase C and calcium channels pathways. The adrenal medulla also presents V1a receptors, but in rat adrenal medulla, only functional V1b receptors have been characterized. Therefore, AVP physiologically regulates adrenal gland functions via autocrine/paracrine mechanisms [42]. In PMAH patients, there is an association between hypercortisolism and abnormal response to AVP. Molecular and pharmacological studies showed eutopic V1a receptor overexpression and/or ectopic V1b and V2 receptors subtypes expression in some PMAH tissues [43e45]. Confirmation of AVP’s involvement in hypercortisolism was detected in PMAH patients after administration of a V1a antagonist, which significantly diminished urinary cortisol levels [46] and in vitro by cell culture experiments [36]. In contrast to normal adrenal glands, PMAH tissues contain two sources of AVP, chromaffin medulla adrenal and steroidogenic cells, and illegitimate adrenal AVP receptors, which are activated by locally produced AVP. Altogether, abnormal AVP production and functional receptors indicate AVP’s involvement in the autocrine/paracrine mechanism of PMAH hypercortisolism.
Intraadrenal production of proopiomelanocortin/ACTH Adrenal medullary tissue expresses corticotropinreleasing hormone (CRH) the gene encoding the precursor of ACTH proopiomelanocortin (POMC) which mediates detectable amounts of ACTH released by chromaffin cells [47e49]. The physiological significance of the corticomedullary interaction through medullary ACTH production remains unclear [50]. PMAH also has ACTH-producing cells first observed by Pereira et al. in 2001 [51]. In a series of 30 PMAH cases expressing ACTH, POMC and the prohormone convertase type 1 were extensively observed, and PMAH steroidogenic cells may generate bioactive ACTH after processing its precursor [29]. ACTHpositive PMAH cells were shown to express gonadal markers consistent with a previous report showing POMC and ACTH expression in gonadal cells [52], suggesting abnormal differentiation and/or separation of the adrenogonadal primordium in PMAH. www.sciencedirect.com
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In vivo, adrenal nodule ACTH secretion was confirmed by ACTH gradient values from adrenal vein sampling in two patients with PMAH compared with the ACTH from peripheral blood samples [28] whereas in vitro ACTH release was irregular with preserved rhythmicity in agreement with observations in PMAH patients [53]. ACTH upregulates MC2R and POMC mRNA in normal adrenal and in PMAH tissue [54]. Therefore, ACTHsecreting cells are positive for MC2R in PMAH tissue indicating that intraadrenal ACTH may exert autocrine actions during cortisol production [29].
Adrenaline and noradrenaline from the adrenal medulla Adrenaline and noradrenaline are produced by the adrenal medulla under control of the splanchnic nerve and cytokines. In bovine adrenal cell cultures, catecholamines induced glucocorticoid production [55]. Healthy human adrenal slices displayed an aldosterone, but not a cortisol, secretory response to noradrenaline [56]. Therefore, there is no consistent evidence for adrenaline and noradrenaline affecting cortisol production in human adrenocortical cells. However, in hyperplasic tissue, clusters of chromogranin A-positive chromaffin cells were described in contact with steroidogenic cells [29]. In addition, PMAH patients were described with abnormal cortisol secretion by catecholamines in two members of the same family [44]. More recently, a study in hyperplasic tissues of members of same family with ARMC5 mutated-PMAH exhibited expression of b1- or b2-adrenergic receptors efficiently coupled to steroidogenesis [56]. The increase and decrease of plasma cortisol concentrations in response to beta-adrenergic receptor agonists and betablockers, respectively, provides evidences of adrenergic control of steroidogenesis in PMAH [57e59]. In summary, these data suggest a positive regulatory adrenergic activity, which contributes to pathological hypercortisolism in PMAH.
Leptin Leptin modulates both the hypothalamic-pituitaryadrenal axis and the sympathetic-adrenomedullary system, regulating energy balance and body weight. Leptin was demonstrated to negatively regulate cortisol production stimulated by ACTH in the human adrenal glands without influencing P45017a or P450SCC mRNA expression or cell proliferation [60]. In contrast, an abnormal stimulatory effect of leptin on cortisol secretion was detected in PMAH cultured cells derived from a patient with food-dependent Cushing’s syndrome, whereas no stimulation was observed from another patient with PMAH and Cushing’s syndrome that did not depend on food intake and in normal adrenocortical cells [61]. Thus, the adipo-adrenal interaction mediated by leptin in PMAH may act as a Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121
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metabolic signal that participates in the pathophysiology of hypercortisolism.
Genetics of PMAH Pathogenic allelic variants, gene duplications, and high frequency of rare missense variants have been identified in the genes firstly associated with PMAH, such as MC2R (melanocortin 2 receptor), GNAS (encoding the stimulatory G protein alpha subunit), MEN1 (multiple endocrine neoplasiatype1), APC (adenomatous polyposis coli), PDE11A (phosphodiesterase 11A), FH (fumarate hydratase), and PRKACA (catalytic subunit alpha of protein kinase A) (Table 1). However, all these genetic defects mentioned associated with PMAH, explained only a few cases. The armadillo repeat-containing 5 (ARMC5) gene identified in 2013 [61], as new gene in PMAH, represents the mainly genetic cause of PMAH once it is frequently mutated in PMAH patients (55%) [62]. Subsequently, several groups have reported germline and somatic mutations of the suppressor gene, ARMC5 in PMAH-patients with variable frequencies ranging from 20% to 26% [63e68]. Interestingly, clinical presentation of pathogenic germline’s carriers is not homogeneous and range from subclinical, mild hypercortisolism to overt Cushing’s syndrome, even in the same family. Collaborative studies involving a large number of patients will provide more consistent data on genotype-phenotype correlations.
Cell culture of PMAH cells—management and potential for using PMAH cultures in functional and translational studies
PMAH is primarily genetically determined by ARMC5, but the mechanisms by which ARMC5 favors the
development of hyperplasia/hypercortisolism are unknown. Identification of autocrine/paracrine regulatory mechanisms is helpful for understanding the physiology and pathological processes in response to ARMC5. These autocrine/paracrine regulatory events, which were summarized in this review, have been detected in vivo in PMAH patients or by complementary studies in vitro using hyperplasia tissue as dispersed cells in culture. In vitro cell cultures from normal and pathologic adrenal cortex have been an important tool for the study of molecular and cellular mechanisms of the adrenal gland [67]. Moreover, primary cultures of adrenal cortex cells have been key in revealing many molecular and cellular aspects of adrenal cortex function [69,70]. In particular, human adrenocortical cell cultures are crucial as a model for molecular and cellular studies that cannot be performed in humans or animals. Herein, we discuss the use of human cell cultures from nodules of primary macronodular adrenocortical hyperplasia to understand the role of ARMC5 and the faulty autocrine/paracrine loops identified. The first report of a functional ARMC5 was performed in an adrenocortical carcinoma cell line, H295R cells [62]. Overexpression of ARMC5 reportedly led to cell death, whereas ARMC5 knockout decreased MC2R expression, steroidogenic enzyme levels, and cortisol production. The H295R cell line represents the most widely used cell culture but is not necessarily predictive of actual PMAH behavior. As such, it was necessary to identify more suitable biological models to properly represent PMAH pathophysiology. For this purpose, cell cultures were obtained from the adrenal nodules of 13 patients diagnosed with PMAH with Cushing’s syndrome (69,2%) and subclinical Cushing’s syndrome established according to clinical, laboratory, and radiological data (Fig. 1). Genetic investigation identified germline
Table 1 The main genes associated with PMAH. Gene
Locus
Role
MC2R GNAS MENIN
18p11 20q13 5q12-22
APC FH PRKACA PDE11A ARMC5
1q42 19p13.1
Regulation of cortisol secretion and adrenal growth Stimulation of adenyl cyclase, cAMP/PKA pathway activation Regulation of gene transcription, cell proliferation-differentiation, apoptosis, and genomic stability Prevention of B-catenin accumulation Involved in Krebs cycle and amino acid metabolism Activation of the cAMP/PKA pathway Hydrolysis of cAMP and cGMP Potential role in apoptosis and steroidogenesis
2q31-35 16p11
Genetic alteration Germline mutations Somatic mutations (early postzygotic event) Germline mutations Germline mutations Germline mutations Germline duplication High frequency of rare germline allelic variants Germline and somatic mutations
MC2R, melanocortin 2 receptor; GNAS, encoding the stimulatory G protein alpha subunit; MEN1, multiple endocrine neoplasiatype1; APC, adenomatous polyposis coli; PDE11A, phosphodiesterase 11A; FH, fumarate hydratase; PRKACA, catalytic subunit alpha of protein kinase A; PMAH, Primary macronodular adrenal hyperplasia
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mutations in the ARMC5 gene in eight of 13 patients, five of whom were carriers of a second event, somatic mutation, or loss of heterozygosity. Cell cultures obtained from the adrenal nodules of PMAH patient’s, carriers or noncarriers of ARMC5 germline mutations, were first morphologically, functionally, and molecularly characterized by Cavalcante et al. [71]. Up to now, in vitro human adrenal cortex research has largely depended on the use of a few immortalized cell lines, H295R [72,73], even though the latter lacks a steroidogenic phenotype, and the utility of this cell line as an adrenocortical model system is questioned. These cell line models are commercially available and have the advantage of large amounts of material and experimental replications. In addition, these lines ensure the conservation of molecular and genetic features. However, these human adrenocortical cell lines also have limitations for the study of PMAH. These cell line cultures do not faithfully reproduce the molecular crosstalk and the cell-cell interactions, for example, between spongiocytes and compact cells that contribute to the PMAH microenvironment. Moreover, this result in a gap in our understanding of pathway modulations that occurs during PMAH development. Use of primary PMAH cultures is dependent on the availability of surgical material, and the pathologist is key in this process because of their expertise in the histological and molecular classification of PMAH. There are a number of protocols outlining the management of patient material for basic and preclinical studies [74].
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Using a specific protocol for surgical PMAH nodules dissection, tissue nodules dissociation is the first step to obtain a single-cell suspension, which can be done using mechanical and enzymatic processes. This is achieved by disaggregation of the tissue into small fragments using a sterile scalpel or scissors. This is followed by enzymatic disaggregation using specific and selected enzymes previously tested to obtain the best cellular viability and yield. Owing to the heterogeneity of PMAH nodules presentation, enzymatic concentration and exposure times depend on the amount of adipose tissue, vessels, and extracellular matrix, as well as on fragment size. The number of cells obtained depends on the amount and condition of surgical tissue available. As such, there is still no standard protocol for obtaining cells from PMAH nodules. Critical management of cell cultures from PMAH involves preservation of the microenvironment following the use of early passages. A small number of passages does not generally cause phenotypic alterations in PMAH cell cultures but does drastically impact the steroidogenic function of PMAH cells (results not published). Accordingly, all experiments should be conducted with PMAH cells below passage fourth. Indeed, early cell passaging leads to changes in gene expression and proliferation rates, affecting the value of cell culture models. Thus, the first steps of cell culture manipulation are of prime importance if useful results are to be achieved [75]. The last step in this process is the morphological characterization of the PMAH cell culture, which is needed to validate the cell phenotype and to exclude crosscontamination by other cells [76]. For this purpose, all
Figure 1
Representative photomicrograph of (a) nonmutated and (b) mutated primary macronodular adrenal hyperplasia cell-cultures.
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PMAH cell cultures are authenticated using short tandem repeat analysis. To confirm that cell cultures obtained are representative of the nodules extracts from patients, cell culture sequencing is performed to detect the presence or absence of ARMC5 mutations and compared with the sequence obtained from PMAH patient blood samples. Functional and molecular characterization of PMAH cell cultures is achieved through detection of lipid droplets in the cytoplasm, reduced expression of steroidogenic enzymes, and a wide variety of illicit paracrine factors and receptors, such as POMC, intraadrenal ACTH, MC2R, AVPR1a/AVPR2, 5HT4R, and GIPR, which are likely involved in hypercortisolism [30]. Functionally, nonmutated PMAH cell cultures stimulated with ACTH exhibit upregulation of POMC mRNA expression, suggesting an autocrine regulatory mechanism triggered by the intracrine ACTH as proposed by Louiset et al. [29]. After 6 h, ACTH, increased StAR, CYP11A1, CYP17A1, and MC2R mRNA expression, demonstrating that in PMAH, as in normal adrenal cells, ACTH increases steroidogenesis and enhances accumulation of its own receptor [54,77]. After 48 h ACTH exposure, increased ACTH expression was observed, demonstrating ACTH’s capacity to upregulate its own expression and suggesting that intraadrenal ACTH secretion is not stimulated by aberrant receptors alone. ARMC5 silencing in PMAH cell cultures illustrated the capacity of ARMC5 to control the cell cycle owing to increase of CCNE1, a cyclin required for G1/S transition [79] and proliferation control. Recently, it was shown that CUL3, a protein involved in the ubiquitinproteasome system, targets ARMC5 for proteasomal degradation [80]. Interesting, Hu et al. in 2017 [81] showed that ARMC5 has a vital functions in fetal development, T cell biology immune response, and adrenal gland biology in mice. ARMC5 can have opposing functions depending on the cell type and conditions, reinforcing the need for appropriate experimental models for the molecular, cellular, and function studies of adrenal macronodular hyperplasia.
Conclusion The gold standard for molecular and cellular research of human adrenal neoplasia is adrenal cortex cell line experimentation. Closer collaboration between clinicians and researchers, along with improved laboratory methodological approaches, have led to hyperplasia, human adrenal normal, and tumor cell cultures becoming a promising new option for this area of research. This cell system maintains the original phenotype of the lesion preserving the original features that are important for reproduction of the tumor microenvironment. This feature allows more reliable studies for identification of autocrine/paracrine Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121
regulatory mechanisms and will facilitate understanding of the physiological and pathological process of ARMC5and its partners, potentially leading to the identification of therapeutic targets in immune disorders and PMAH.
Conflict of interest statement Nothing declared.
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The present study showed that in patient with primary macronodular adrenal hyperplasia and Cushing’s syndrome, cortisol production is controlled both by aberrant membrane receptors and ACTH that is produced within the adrenoctical nodules, amplifing the action of abnormal receptors. 30. Lefebvre H, Duparc C, Prévost G, Bertherat J, Louiset E: Cell-tocell communication in bilateral macronodular adrenal hyperplasia causing hypercortisolism. Front Endocrinol (Lausanne) 2015, 6:34. 31. Lefebvre H, Contesse V, Delarue C, Feuilloley M, Hery F, Grise P, et al.: Serotonin-induced stimulation of cortisol secretion from human adrenocortical tissue is mediated through activation of a serotonin4 receptor subtype. Neuroscience 1992, 47:999–1007. 32. Lefebvre H, Compagnon P, Contesse V, Delarue C, Thuillez C, Vaudry H, et al.: Production and metabolism of serotonin (5HT) by the human adrenal cortex: paracrine stimulation of aldosterone secretion by 5-HT. J Clin Endocrinol Metab 2001, 86:5001–5007. 33. Cartier D, Jégou S, Parmentier F, Lihrmann I, Louiset E, Kuhn JM, et al.: Expression profile of serotonin4 (5-HT4) receptors in adrenocortical aldosterone-producing adenomas. Eur J Endocrinol 2005, 153:939–947.
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23. Swords FM, Noon LA, King PJ, Clark AJ: Constitutive activation of the human ACTH receptor resulting from a synergistic interaction between two naturally occurring missense mutations in the MC2R gene. Mol Cell Endocrinol 2004, 213: 149–154. 24. Weinstein LS, Shenker A, Gejman PV, Merino MJ, Friedman E, Spiegel AM: Activating mutations of the stimulatory G protein in the McCune-Albright syndrome. N Engl J Med 1991, 325: 1688–1695. 25. Fragoso MC, Domenice S, Latronico AC, Martin RM, Pereira MA, * Zerbini MC, et al.: Cushing’s syndrome secondary to adrenocorticotropin-independent macronodular adrenocortical hyperplasia due to activating mutations of GNAS1 gene. J Clin Endocrinol Metab 2003, 88:2147–2151. This work showed the association of two different gsp somatic mutations at codon ARg201 in three out five patients with PMAH. All of them did not echibit any signs of McCune Albright syndrome. 26. Hsiao HP, Kirschner LS, Bourdeau I, Keil MF, Boikos SA, Verma S, et al.: Clinical and genetic heterogeneity, overlap with other tumor syndromes, and atypical glucocorticoid hormone secretion in adrenocorticotropin-independent macronodular adrenal hyperplasia compared with other adrenocortical tumors. J Clin Endocrinol Metab 2009, 94: 2930–2937. 27. Horvath A, Boikos S, Giatzakis C, Robinson-White A, Groussin L, Griffin KJ, et al.: A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 (PDE11A) in individuals with adrenocortical hyperplasia. Nat Genet 2006, 38: 794–800. 28. Cao Y, He M, Gao Z, Peng Y, Li Y, Li L, et al.: Activating hotspot L205R mutation in PRKACA and adrenal Cushing’s syndrome. Science 2014, 344:913–917. 29. Louiset E, Duparc C, Young J, Renouf S, Tetsi Nomigni M, * Boutelet I, et al.: Intraadrenal corticotropin in bilateral macronodular adrenal hyperplasia. N Engl J Med 2013, 369: 2115–2125.
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blockade therapy and interest of unilateral adrenalectomy. Endocr J 2009, 56:867–877. 60. Glasow A, Bornstein SR: Leptin and the adrenal gland. Eur J Clin Investig 2000, 30:39–45. 61. Pralong FP, Gomez F, Guillou L, Mosimann F, Franscella S, Gaillard RC: Food-dependent Cushing’s syndrome: possible involvement of leptin in cortisol hypersecretion. J Clin Endocrinol Metab 1999, 84:3817–3822. 62. Assié G, Libé R, Espiard S, Rizk-Rabin M, Guimier A, Luscap W, * et al.: ARMC5 mutations in macronodular adrenal hyperplasia with Cushing’s syndrome. N Engl J Med 2013, 369:2105–2114. The authors, using genomic studies identified germiline ARMC5 mutations in a substantial proportion of patients with PMAH. After this finding and increas number of familial and apparently sporadic cases has been described in literature. 63. Elbelt U, Trovato A, Kloth M, Gentz E, Finke R, Spranger J, et al.: * Molecular and clinical evidence for an ARMC5 tumor syndrome: concurrent inactivating germline and somatic mutations are associated with both primary macronodular adrenal hyperplasia and meningioma. J Clin Endocrinol Metab 2015, 100:E119–E128. The authors proved an additional role for ARMC5 as the development for intracranial menigiomas in one patient with PMAH, previously observed by Alencar G et al. ref 68. 64. Espiard S, Drougat L, Libé R, Assié G, Perlemoine K, Guignat L, et al.: ARMC5 mutations in a large cohort of primary macronodular adrenal hyperplasia: clinical and functional consequences. J Clin Endocrinol Metab 2015, 100:E926–E935. 65. Faucz FR, Zilbermint M, Lodish MB, Szarek E, Trivellin G, Sinaii N, et al.: Macronodular adrenal hyperplasia due to mutations in an armadillo repeat containing 5 (ARMC) gene: a clinical and genetic investigation. J Clin Endocrinol Metab 2014, 99:E1113–E1119. 66. Drougat L, Espiard S, Bertherat J: Genetics of primary bilateral macronodular adrenal hyperplasia: a model for early diagnosis of Cushing’s syndrome? Eur J Endocrinol 2015, 173: M121–M131. 67. De Venanzi A, Alencar GA, Bourdeau I, Fragoso MC, Lacroix A: Primary bilateral macronodular adrenal hyperplasia. Curr Opin Endocrinol Diabetes Obes 2014, 21:177–184. 68. Alencar GA, Lerario AM, Nishi MY, Mariani BM, Almeida MQ, * Tremblay J, et al.: ARMC5 mutations are a frequent cause of primary macronodular adrenal hyperplasia. J Clin Endocrinol Metab 2014 Aug, 99:E1501–E1509, https://doi.org/10.1210/ jc.2013-4237. The present study demonstrated that inherited autossomal dominant mutations in the ARMC5 gene are a frequent cause of familial patients with PMAH. 69. Wang T, Rainey WE: Human adrenocortical carcinoma cell lines. Mol Cell Endocrinol 2012, 351:58–65. 70. Mattos GE, Jacysyn JF, Amarante-Mendes GP, Lotfi CF: Comparative effect of FGF2, synthetic peptides 1-28 N-POMC and ACTH on proliferation in rat adrenal cell primary cultures. Cell Tissue Res 2011, 345:343–356. 71. França MM, Abreu NP, Vrechi TA, Lotfi CF: POD-1/Tcf21 overexpression reduces endogenous SF-1 and StAR expression in rat adrenal cells. Braz J Med Biol Res 2015, 48:1087–1094. 72. Cavalcante IP, Nishi M, Zerbini MCN, Almeida MQ, Brondani VB, * Botelho MLAA, et al.: The role of ARMC5 in human cell cultures from nodules of primary macronodular adrenocortical hyperplasia (PMAH). Mol Cell Endocrinol 2018, 460:36–46. In this work, the authors confirmed the role of ARMC5 as an important pro-apoptotic protein involved in PMAH-related steroidogenesis. In addition, they repoted for the first time in a sutiable model of PMAHculture cells that ARMC5 is involved in cotrol of cell proliferation and in cell cycle regulation. 73. Gazdar AF, Oie HK, Shackleton CH, Chen TR, Triche TJ, Myers CE, et al.: Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res 1990, 50:5488–5496.
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80. E C, A V, L D, C L, MCBV F, M R-R, et al IP C: Cullin 3 is a * partner of Armadillo repeat containing 5 (ARMC5), the product of the gene responsible for primary bilateral macronodular adrenal hyperplasia. In Cullin 3 and ARMC5. Annales d’Endocrinology2018; 2019:184–185. Endocrine Abstracts, https://www.endocrine-abstracts.org/ea0063oc10.4; 2019. 63 OC 10.4 DOI: 10.1530/endoabs.63.OC10.4. In this study, the authors demonstrated for the first time that the interaction between ARMC5 and CUL3 leads to the ubiquitination of ARMC5, suggesting that ARMC5 is a substrate for a CUL3-based ubiquitin complex. These data show a new mechanism of regulation of the ARMC5 protein and open new perspectives for understanding the pathophysiology of PBMAH. 81. Hu Y, Lao L, Mao J, Jin W, Luo H, Charpentier T, et al.: Armc5 * deletion causes developmental defects and compromises Tcell immune responses. Nat Commun 2017, 8:13834. Animal model study using Armc5 knockout mice, indicated that ARMC5 is crucial in fetal development, T-cell function and adrenal gland growth homeostasis, and that the functions of ARMC5 probably depend on interaction with multiple signaling pathways.
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Current Opinion in
Endocrine and Metabolic Research
Reviews
Molecular and cellular regulation of primary macronodular adrenal hyperplasia Maria Candida Barisson Villares Fragoso1 and Claudimara Ferini Pacicco Lotfi2 Abstract
Available online 21 August 2019
Primary macronodular adrenal hyperplasia (PMAH) is an adrenal cause of Cushing’s syndrome, the clinical and the pathological features of which were described for the first time by Marvin Kirschner et al. in 1964 [1]. At that time, the authors suggested the following as an explanation for this heterogeneous adrenal disorder: a more tenable hypothesis would be that multinodular hyperplasia with macronodules represents an abnormal outcome of adrenal stimulation, with eventual concomitant, possibly localized, loss of adrenal secretory efficiency. Over the 55 years since PMAH’s identification, our knowledge has advanced to clarify the mechanisms involved in the pathophysiology of this disorder. Up to now, diverse molecular events have been proposed to explain the enhanced cortisol secretion, cell proliferation, and development of macronodules that occur in PMAH. Nevertheless, the precise sequence of events and molecular mechanisms underlying in this condition remaining incompletely elucidated. Recent studies indicate that PMAH is more genetically determined than previously thought according to the bilateral nature of the disease. In addition, the description of familial forms, with an autosomal dominant pattern suggested an inherited genetic etiology for PMAH. The purpose of this review is to summarize the primary insights concerning the molecular and cellular regulatory mechanisms of PMAH.
https://doi.org/10.1016/j.coemr.2019.08.007
Addresses 1 Unidade de Suprarrenal, Disciplina de Endocrinologia e Metabologia, Hospital Das Clinicas da Faculdade de Medicina da USP, Instituto Do Câncer Do Estado de São Paulo – ICESP, University of Sao Paulo, Brazil 2 Instituto de Ciências Biomédicas Do Departamento de Anatomia, University of Sao Paulo, Brazil Corresponding author: Barisson Villares Fragoso, Maria Candida ([email protected])
Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121 This review comes from a themed issue on Adrenal Cortex Edited by André Lacroix and Enzo Lalli For a complete overview see the Issue and the Editorial
Funding statement from Fundac¸˜ao de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP) to MCBVF # 2015/50192-9. Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121
2451-9650/© 2019 Elsevier Ltd. All rights reserved.
Keywords Adrenal hyperplasia, PMAH, Cushing’s syndrome, Pathophysiology.
Introduction Primary macronodular adrenal hyperplasia (PMAH) was first described by Marvin Kirschner et al. in 1964 [1] and it is considered a rare cause of endogenous hypercortisolism occurring in less than 2% of all cases of Cushing’s syndrome. Nevertheless, it has been more frequently diagnosed than in the past because of increasing of radiological abdominal exams showing adrenal incidentaloma and because of the recognition of genetic inheritance in more than 50% of patients. Few years ago, inactivating germline and somatic mutations in the armadillo repeat-containing 5 (ARMC5) gene was accepted as a major genetic cause of patients with PMAH.
Cellular characteristics PMAH Macroscopic examination of the macronodular adrenal hyperplasia shows characteristic increase in adrenal mass, which can reach up to 100 times the normal weight and small to large nodules with yellow coloration [2]. Microscopically, this presents as nonpigmented nodules composed of two groups of cell types with no internodular atrophy [3]. Spongiocytes group of cells with clear lipid-rich cytoplasmic regions are a predominant component of the nodules (personal information), with compact cells forming an island-like structure with lipid-poor cytoplasmic regions. No mitosis or nuclear variability with respect to size and shape is observed in either cell types. Immunolocalization of steroidogenic enzymes in both cell types showed 3b-hydroxysteroid dehydrogenase/D5 4 isomerase distribution within spongiocytes, whereas the steroid 17 alpha-hydroxylase/ 17e20 lyase (P450c17) is detected in compact cells [4]. The inefficient steroidogenesis in this celleenzymatic profile could in part explain the mild hypercortisolism observed in PMAH patients despite enlargement of the adrenal glands [5]. www.sciencedirect.com
Molecular and cellular regulation of PMAH Fragoso and Lotfi
Aberrant G-protein-coupled membrane receptors Under physiological condition, adrenocorticotropic hormone (ACTH) is the primary regulator of the steroidogenesis. ACTH acts through of its melanocortin 2 receptor (MC2R), a G protein-coupled receptor (GPCR). This complex hormone-receptor activation determines cAMP/protein kinase A (PKA) pathway stimulation and increases steroidogenesis and cell proliferation in the adrenal cortex [5]. The presence of aberrant ectopic or eutopic hormone receptors excessively expressed in the adrenal cells in PMAH, despite suppressed ACTH levels, present the capacity to stimulate steroidogenesis and has been recognized as part of the pathophysiology of PMAH [6]. Several GPCRs have been described in PMAH nodules such as the receptor for luteinizing hormone (LH)/ human chorionic gonadotropin (hCG), the serotonin receptor (5-HT4 and 5-HT7 receptors), the leptin receptor, glucose-dependent insulinotropic peptide (GIP), catecholamine (b-adrenergic receptor), vasopressin (V1eV2eV3e vasopressin receptor), glucagon, and angiotensin II. However, whether high GPCR expression and their response to specific ligands is a primary event or secondary phenomenon resulting from cell proliferation and dedifferentiation is not entirely clear [6,7].
GIP and glucagon effects in PMAH cells Hamet et al. in 1987 proposed a humoral factor induced by eating as responsible for periodic adrenal cortisol secretion [8] who observed that plasma fasting cortisol was low in the morning and increase following meal ingestion. This finding subsequently confirmed by Lacroix et al. in 1992 [9]. In vivo physiological concentrations of GIP stimulate cortisol production in PMAH patients in whom the presence of GIP receptors was suggested by adrenal imaging after injection of [123I]GIP. In dispersed cells from PMAH patients, GIP mediated cortisol secretion in contrast with normal adult or fetal adrenal cells. In addition, adrenal tissues from patients with GIP-dependent Cushing’s syndrome present overexpressed nonmutated GIP receptors. In normal human adrenal glands, glucose-dependent insulinotropic polypeptide receptor (GIPR) is barely expressed and is not related to efficient steroidogenesis [10]. However, GIP stimulates thymidine incorporation in PMAH adrenal cells but not normal cells [11]. In addition, activation of the GIPR receptor in PMAH patients with GIP-dependent adrenal Cushing’s syndrome is functionally coupled to cAMP signaling and triggers adrenal cell proliferation [6]. More recently, the molecular pathogenesis of GIP-dependent Cushing’s syndrome was shown to occur through monoallelic transcriptional activation of GIPR driven by chromosome 19q13 rearrangements [12].
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Together with GIP and others structurally related peptide hormones (glucagon-like peptides, vasoactive intestinal peptide, pituitary adenylyl cyclase-activating polypeptide, and growth hormone-releasing hormone) glucagon activates receptors coupled to the cAMP/PKA pathway [13]. The early observation that adenylyl cyclase activity is enhanced by glucagon in PMAH [14] led to hypothesis that illegitimate glucagon receptors are associated with Cushing’s syndrome. This finding was supported by an abnormal plasma cortisol response to glucagon in 58% of patients with PMAH as well as subclinical hypercortisolism [15]. More recently, evaluation of glucagon’s role in the regulation of cortisol secretion in PMAH patients has revealed the presence of glucagon receptor-like immunoreactivity in clusters of spongiocytes in adrenal tissues from patients who were sensitive in vivo to glucagon [16].
LH and LHCGR effects In 1999, Lacroix et al. [17]described a patient with Cushing’s syndrome because of PMAH with cortisol production stimulated in vivo by LH and hCG during her pregnancies, which became constant only after menopause. This suggests a functional LHCG receptor coupled to steroidogenesis. This receptor activates adenylyl cyclase and phospholipase C to stimulate gonadal steroidogenesis and the zona reticularis in the human adrenal cortex [18], whereas hCG stimulates the secretion of dehydroepiandrosterone sulphate in human fetal adrenal cells [19]. In this case, suppression of endogenous levels of LH with leuprolide acetate was able to normalize cortisol production. In mice, gonadectomy induces hyperplasia and tumorigenesis, apparently triggered by elevated postgonadectomy levels of LH/hCG, followed by ectopic upregulation of Luteinizing hormone/choriogonadotropin receptor (LHCGR). However, gonadectomy in tumorigenic, DBA/2J, and nontumorigenic C57BL/6J mice induced similar upregulation of adrenal LHCGR in both indicating that ectopic expression of this receptor is not the direct cause of tumors [20]. Similarly, analysis of aberrant LHCGR expression in different human adrenal hyperplasia patients (7 with PMAH, 5 with primary pigmented nodular adrenocortical disease and 8 with diffuse adrenal hyperplasia secondary to Cushing’s disease ) showed no differences between LHCGR expression among all tissues examined [21]. Interesting, there is a description of two cases with PMAH whose adrenal lesions expressed LHCGR and who had response to leuprolide acetate treatment even in the absence of increased cortisol. In addition, a study described novel features of gene expression LHCGR-mediated, pregnancy-induced through mechanism mutation-independent by hCG activated. In this study, a young prim-gravid patient with over Cushing’s syndrome within a week after delivery, the signs and symptoms of Cushing’s syndrome regressed, and they recovered during a new pregnancy [22]. Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121
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Taken together whole, the data above indicate that circulating hormone levels may influence cortisol release and contribute to the hypercortisolism observed in PMAH.
Activation of the cAMP/PKA pathway Activation of the cAMP-PKA pathway is the central mechanisms associated with PMAH. ACTH acts as a regulator of steroidogenesis and adrenal proliferation through the melanocortin 2 receptor, MC2R [23]. Activating mutations in the conserved MC2R gene lead to constitutive activation of the cAMP-PKA pathway, which controls the autonomy of adrenal gland; however, this mechanism is considered an extremely rare cause of PMAH [23]. Heterotrimeric G protein in the active state can modulate the activity of the ligands and their 7M receptors, increasing cAMP-PKA pathway activation. Somatic mutations of the gene encoding the alpha subunit of stimulatory G protein, GNAS, result in constitutive activation of adenylate cyclase in specific codons. These so-called gsp somatic mutations were previously described in patients with McCune-Albright syndrome associated with Cushing’s syndrome [24]. Interesting, these gsp mutations have also been described in adrenal nodules from PMAH patients, and while considered, a rare molecular event is sufficient to induce adrenal hyperplasia and cortisol secretion [25,26]. Phosphodiesterase’s (PDE) enzymes families are considered key regulators of intracellular cAMP levels. Recent studies have shown that the presence of inactivating germline mutations and allelic variants in PDE11A may increase susceptibility to PMAH [27]. In HEK293 cell lines, these allelic variants exhibited higher cAMP levels than wild-type cell lines, suggesting that these mutations cause reduced cAMP hydrolytic activity. Somatic activating mutations in the PRKACA gene, which encodes the catalytic subunit alpha PKA, were identified in 40% of adrenal adenomas producing cortisol. Interesting, germline duplications on a genomic region of chromosome 19 that included PRKACA were identified in PMAH patients [28]. Further studies in large cohorts of patients are needed to determine the frequency and effect of these molecular events on the pathogenesis of PMAH.
Paracrine/autocrine regulation of cortisol production in PMAH More recently, the notion has emerged that, in addition to membrane G-protein-coupled receptors, there are Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121
abnormal paracrine/autocrine factors in PMAH tissue distinct from the physiological paracrine/autocrine systems occurring in the normal adrenal gland. These abnormal factors lead to abnormal intraadrenal stimulatory loops and cortisol hypersecretion [29]. From studying, autocrine/paracrine/regulation of cortisol secretion in PMAH, a new concept of paracrinopathy emerged that was proposed in 2015 by Lefebvre et al. [30]. Some of the important autocrine/paracrine regulatory signals of cortisol secretion in PMAH are discussed in more detail below.
Serotonin In the normal human adrenal gland, 5hydroxytryptamine (5-HT) is produced and released by mast cells located in the subcapsular adrenal region, which stimulates corticosteroids secretion through the activation of 5-HT4 receptor positively coupled to adenylyl cyclase and calcium influx [31,32]. In situ hybridization in the normal adrenal cortex revealed that 5HT4 receptor mRNA was expressed in the zona glomerulosa, and a weaker hybridization signal was also visualized in zona fasciculata/reticularis cells [33,34]. In vitro studies demonstrated that 5-HT is efficient at stimulating aldosterone but is less efficient at stimulating cortisol [35]. In agreement with in vitro studies, administration of 5-HT4 receptor agonists in healthy patient induced increased aldosterone levels without affecting cortisol levels [35]. Distinct from normal adrenal glands, where the source of 5-HT is mast cells, immunohistochemistry analysis in PMAH tissues revealed abnormal synthesis of 5-HT in clusters of steroidogenic cells, suggesting an autocrine/ paracrine mechanism for cortisol production by 5-HT [36]. Clinical studies and analysis in PMAH tissues reported over expression of the 5-HT4 receptor, indicating mediation of the 5-HT4 receptor by serotonin’s action [37,38]. However, in some PMAH tissues, eutopic 5HTR4 expression was found in addition to ectopic expression of 5HTR7 receptor in the plasma membrane of steroidogenic cells. Both receptors are positively coupled with adenylyl cyclase, mediating corticosteroid-induced 5HT in PMAH through the cAMP/PKA pathway [39]. More recently, a study showed that in PMAH, high expression of TPH1 (5-HT synthesizing enzyme tryptophan hydroxylase) and 5HT4R was associated with ACTH-positive cells suggesting ACTH as a regulator of these proteins via an autocrine/paracrine mechanism. In contrast, 5-HT7R seems less dependent on the local synthesis of ACTH [40]. In summary, distinct from the normal adrenal gland, the presence of aberrant 5-HT production and illicit www.sciencedirect.com
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expression of serotonin receptors improve the 5-HT autocrine/paracrine pathway and hypercortisolism in PMAH.
Arginine vasopressin The arginine vasopressin (AVP) released by hypothalamic neurons stimulates ACTH production and glucocorticoid production in the adrenal glands. However, AVP is also released by chromaffin cells from adrenal medulla acting as a paracrine regulator of glucocorticoid production [41]. Vasopressin type 1a (V1a) receptors are present in the adrenal cortex and act via heterotrimeric G proteins phospholipase C and calcium channels pathways. The adrenal medulla also presents V1a receptors, but in rat adrenal medulla, only functional V1b receptors have been characterized. Therefore, AVP physiologically regulates adrenal gland functions via autocrine/paracrine mechanisms [42]. In PMAH patients, there is an association between hypercortisolism and abnormal response to AVP. Molecular and pharmacological studies showed eutopic V1a receptor overexpression and/or ectopic V1b and V2 receptors subtypes expression in some PMAH tissues [43e45]. Confirmation of AVP’s involvement in hypercortisolism was detected in PMAH patients after administration of a V1a antagonist, which significantly diminished urinary cortisol levels [46] and in vitro by cell culture experiments [36]. In contrast to normal adrenal glands, PMAH tissues contain two sources of AVP, chromaffin medulla adrenal and steroidogenic cells, and illegitimate adrenal AVP receptors, which are activated by locally produced AVP. Altogether, abnormal AVP production and functional receptors indicate AVP’s involvement in the autocrine/paracrine mechanism of PMAH hypercortisolism.
Intraadrenal production of proopiomelanocortin/ACTH Adrenal medullary tissue expresses corticotropinreleasing hormone (CRH) the gene encoding the precursor of ACTH proopiomelanocortin (POMC) which mediates detectable amounts of ACTH released by chromaffin cells [47e49]. The physiological significance of the corticomedullary interaction through medullary ACTH production remains unclear [50]. PMAH also has ACTH-producing cells first observed by Pereira et al. in 2001 [51]. In a series of 30 PMAH cases expressing ACTH, POMC and the prohormone convertase type 1 were extensively observed, and PMAH steroidogenic cells may generate bioactive ACTH after processing its precursor [29]. ACTHpositive PMAH cells were shown to express gonadal markers consistent with a previous report showing POMC and ACTH expression in gonadal cells [52], suggesting abnormal differentiation and/or separation of the adrenogonadal primordium in PMAH. www.sciencedirect.com
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In vivo, adrenal nodule ACTH secretion was confirmed by ACTH gradient values from adrenal vein sampling in two patients with PMAH compared with the ACTH from peripheral blood samples [28] whereas in vitro ACTH release was irregular with preserved rhythmicity in agreement with observations in PMAH patients [53]. ACTH upregulates MC2R and POMC mRNA in normal adrenal and in PMAH tissue [54]. Therefore, ACTHsecreting cells are positive for MC2R in PMAH tissue indicating that intraadrenal ACTH may exert autocrine actions during cortisol production [29].
Adrenaline and noradrenaline from the adrenal medulla Adrenaline and noradrenaline are produced by the adrenal medulla under control of the splanchnic nerve and cytokines. In bovine adrenal cell cultures, catecholamines induced glucocorticoid production [55]. Healthy human adrenal slices displayed an aldosterone, but not a cortisol, secretory response to noradrenaline [56]. Therefore, there is no consistent evidence for adrenaline and noradrenaline affecting cortisol production in human adrenocortical cells. However, in hyperplasic tissue, clusters of chromogranin A-positive chromaffin cells were described in contact with steroidogenic cells [29]. In addition, PMAH patients were described with abnormal cortisol secretion by catecholamines in two members of the same family [44]. More recently, a study in hyperplasic tissues of members of same family with ARMC5 mutated-PMAH exhibited expression of b1- or b2-adrenergic receptors efficiently coupled to steroidogenesis [56]. The increase and decrease of plasma cortisol concentrations in response to beta-adrenergic receptor agonists and betablockers, respectively, provides evidences of adrenergic control of steroidogenesis in PMAH [57e59]. In summary, these data suggest a positive regulatory adrenergic activity, which contributes to pathological hypercortisolism in PMAH.
Leptin Leptin modulates both the hypothalamic-pituitaryadrenal axis and the sympathetic-adrenomedullary system, regulating energy balance and body weight. Leptin was demonstrated to negatively regulate cortisol production stimulated by ACTH in the human adrenal glands without influencing P45017a or P450SCC mRNA expression or cell proliferation [60]. In contrast, an abnormal stimulatory effect of leptin on cortisol secretion was detected in PMAH cultured cells derived from a patient with food-dependent Cushing’s syndrome, whereas no stimulation was observed from another patient with PMAH and Cushing’s syndrome that did not depend on food intake and in normal adrenocortical cells [61]. Thus, the adipo-adrenal interaction mediated by leptin in PMAH may act as a Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121
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metabolic signal that participates in the pathophysiology of hypercortisolism.
Genetics of PMAH Pathogenic allelic variants, gene duplications, and high frequency of rare missense variants have been identified in the genes firstly associated with PMAH, such as MC2R (melanocortin 2 receptor), GNAS (encoding the stimulatory G protein alpha subunit), MEN1 (multiple endocrine neoplasiatype1), APC (adenomatous polyposis coli), PDE11A (phosphodiesterase 11A), FH (fumarate hydratase), and PRKACA (catalytic subunit alpha of protein kinase A) (Table 1). However, all these genetic defects mentioned associated with PMAH, explained only a few cases. The armadillo repeat-containing 5 (ARMC5) gene identified in 2013 [61], as new gene in PMAH, represents the mainly genetic cause of PMAH once it is frequently mutated in PMAH patients (55%) [62]. Subsequently, several groups have reported germline and somatic mutations of the suppressor gene, ARMC5 in PMAH-patients with variable frequencies ranging from 20% to 26% [63e68]. Interestingly, clinical presentation of pathogenic germline’s carriers is not homogeneous and range from subclinical, mild hypercortisolism to overt Cushing’s syndrome, even in the same family. Collaborative studies involving a large number of patients will provide more consistent data on genotype-phenotype correlations.
Cell culture of PMAH cells—management and potential for using PMAH cultures in functional and translational studies
PMAH is primarily genetically determined by ARMC5, but the mechanisms by which ARMC5 favors the
development of hyperplasia/hypercortisolism are unknown. Identification of autocrine/paracrine regulatory mechanisms is helpful for understanding the physiology and pathological processes in response to ARMC5. These autocrine/paracrine regulatory events, which were summarized in this review, have been detected in vivo in PMAH patients or by complementary studies in vitro using hyperplasia tissue as dispersed cells in culture. In vitro cell cultures from normal and pathologic adrenal cortex have been an important tool for the study of molecular and cellular mechanisms of the adrenal gland [67]. Moreover, primary cultures of adrenal cortex cells have been key in revealing many molecular and cellular aspects of adrenal cortex function [69,70]. In particular, human adrenocortical cell cultures are crucial as a model for molecular and cellular studies that cannot be performed in humans or animals. Herein, we discuss the use of human cell cultures from nodules of primary macronodular adrenocortical hyperplasia to understand the role of ARMC5 and the faulty autocrine/paracrine loops identified. The first report of a functional ARMC5 was performed in an adrenocortical carcinoma cell line, H295R cells [62]. Overexpression of ARMC5 reportedly led to cell death, whereas ARMC5 knockout decreased MC2R expression, steroidogenic enzyme levels, and cortisol production. The H295R cell line represents the most widely used cell culture but is not necessarily predictive of actual PMAH behavior. As such, it was necessary to identify more suitable biological models to properly represent PMAH pathophysiology. For this purpose, cell cultures were obtained from the adrenal nodules of 13 patients diagnosed with PMAH with Cushing’s syndrome (69,2%) and subclinical Cushing’s syndrome established according to clinical, laboratory, and radiological data (Fig. 1). Genetic investigation identified germline
Table 1 The main genes associated with PMAH. Gene
Locus
Role
MC2R GNAS MENIN
18p11 20q13 5q12-22
APC FH PRKACA PDE11A ARMC5
1q42 19p13.1
Regulation of cortisol secretion and adrenal growth Stimulation of adenyl cyclase, cAMP/PKA pathway activation Regulation of gene transcription, cell proliferation-differentiation, apoptosis, and genomic stability Prevention of B-catenin accumulation Involved in Krebs cycle and amino acid metabolism Activation of the cAMP/PKA pathway Hydrolysis of cAMP and cGMP Potential role in apoptosis and steroidogenesis
2q31-35 16p11
Genetic alteration Germline mutations Somatic mutations (early postzygotic event) Germline mutations Germline mutations Germline mutations Germline duplication High frequency of rare germline allelic variants Germline and somatic mutations
MC2R, melanocortin 2 receptor; GNAS, encoding the stimulatory G protein alpha subunit; MEN1, multiple endocrine neoplasiatype1; APC, adenomatous polyposis coli; PDE11A, phosphodiesterase 11A; FH, fumarate hydratase; PRKACA, catalytic subunit alpha of protein kinase A; PMAH, Primary macronodular adrenal hyperplasia
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mutations in the ARMC5 gene in eight of 13 patients, five of whom were carriers of a second event, somatic mutation, or loss of heterozygosity. Cell cultures obtained from the adrenal nodules of PMAH patient’s, carriers or noncarriers of ARMC5 germline mutations, were first morphologically, functionally, and molecularly characterized by Cavalcante et al. [71]. Up to now, in vitro human adrenal cortex research has largely depended on the use of a few immortalized cell lines, H295R [72,73], even though the latter lacks a steroidogenic phenotype, and the utility of this cell line as an adrenocortical model system is questioned. These cell line models are commercially available and have the advantage of large amounts of material and experimental replications. In addition, these lines ensure the conservation of molecular and genetic features. However, these human adrenocortical cell lines also have limitations for the study of PMAH. These cell line cultures do not faithfully reproduce the molecular crosstalk and the cell-cell interactions, for example, between spongiocytes and compact cells that contribute to the PMAH microenvironment. Moreover, this result in a gap in our understanding of pathway modulations that occurs during PMAH development. Use of primary PMAH cultures is dependent on the availability of surgical material, and the pathologist is key in this process because of their expertise in the histological and molecular classification of PMAH. There are a number of protocols outlining the management of patient material for basic and preclinical studies [74].
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Using a specific protocol for surgical PMAH nodules dissection, tissue nodules dissociation is the first step to obtain a single-cell suspension, which can be done using mechanical and enzymatic processes. This is achieved by disaggregation of the tissue into small fragments using a sterile scalpel or scissors. This is followed by enzymatic disaggregation using specific and selected enzymes previously tested to obtain the best cellular viability and yield. Owing to the heterogeneity of PMAH nodules presentation, enzymatic concentration and exposure times depend on the amount of adipose tissue, vessels, and extracellular matrix, as well as on fragment size. The number of cells obtained depends on the amount and condition of surgical tissue available. As such, there is still no standard protocol for obtaining cells from PMAH nodules. Critical management of cell cultures from PMAH involves preservation of the microenvironment following the use of early passages. A small number of passages does not generally cause phenotypic alterations in PMAH cell cultures but does drastically impact the steroidogenic function of PMAH cells (results not published). Accordingly, all experiments should be conducted with PMAH cells below passage fourth. Indeed, early cell passaging leads to changes in gene expression and proliferation rates, affecting the value of cell culture models. Thus, the first steps of cell culture manipulation are of prime importance if useful results are to be achieved [75]. The last step in this process is the morphological characterization of the PMAH cell culture, which is needed to validate the cell phenotype and to exclude crosscontamination by other cells [76]. For this purpose, all
Figure 1
Representative photomicrograph of (a) nonmutated and (b) mutated primary macronodular adrenal hyperplasia cell-cultures.
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PMAH cell cultures are authenticated using short tandem repeat analysis. To confirm that cell cultures obtained are representative of the nodules extracts from patients, cell culture sequencing is performed to detect the presence or absence of ARMC5 mutations and compared with the sequence obtained from PMAH patient blood samples. Functional and molecular characterization of PMAH cell cultures is achieved through detection of lipid droplets in the cytoplasm, reduced expression of steroidogenic enzymes, and a wide variety of illicit paracrine factors and receptors, such as POMC, intraadrenal ACTH, MC2R, AVPR1a/AVPR2, 5HT4R, and GIPR, which are likely involved in hypercortisolism [30]. Functionally, nonmutated PMAH cell cultures stimulated with ACTH exhibit upregulation of POMC mRNA expression, suggesting an autocrine regulatory mechanism triggered by the intracrine ACTH as proposed by Louiset et al. [29]. After 6 h, ACTH, increased StAR, CYP11A1, CYP17A1, and MC2R mRNA expression, demonstrating that in PMAH, as in normal adrenal cells, ACTH increases steroidogenesis and enhances accumulation of its own receptor [54,77]. After 48 h ACTH exposure, increased ACTH expression was observed, demonstrating ACTH’s capacity to upregulate its own expression and suggesting that intraadrenal ACTH secretion is not stimulated by aberrant receptors alone. ARMC5 silencing in PMAH cell cultures illustrated the capacity of ARMC5 to control the cell cycle owing to increase of CCNE1, a cyclin required for G1/S transition [79] and proliferation control. Recently, it was shown that CUL3, a protein involved in the ubiquitinproteasome system, targets ARMC5 for proteasomal degradation [80]. Interesting, Hu et al. in 2017 [81] showed that ARMC5 has a vital functions in fetal development, T cell biology immune response, and adrenal gland biology in mice. ARMC5 can have opposing functions depending on the cell type and conditions, reinforcing the need for appropriate experimental models for the molecular, cellular, and function studies of adrenal macronodular hyperplasia.
Conclusion The gold standard for molecular and cellular research of human adrenal neoplasia is adrenal cortex cell line experimentation. Closer collaboration between clinicians and researchers, along with improved laboratory methodological approaches, have led to hyperplasia, human adrenal normal, and tumor cell cultures becoming a promising new option for this area of research. This cell system maintains the original phenotype of the lesion preserving the original features that are important for reproduction of the tumor microenvironment. This feature allows more reliable studies for identification of autocrine/paracrine Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121
regulatory mechanisms and will facilitate understanding of the physiological and pathological process of ARMC5and its partners, potentially leading to the identification of therapeutic targets in immune disorders and PMAH.
Conflict of interest statement Nothing declared.
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80. E C, A V, L D, C L, MCBV F, M R-R, et al IP C: Cullin 3 is a * partner of Armadillo repeat containing 5 (ARMC5), the product of the gene responsible for primary bilateral macronodular adrenal hyperplasia. In Cullin 3 and ARMC5. Annales d’Endocrinology2018; 2019:184–185. Endocrine Abstracts, https://www.endocrine-abstracts.org/ea0063oc10.4; 2019. 63 OC 10.4 DOI: 10.1530/endoabs.63.OC10.4. In this study, the authors demonstrated for the first time that the interaction between ARMC5 and CUL3 leads to the ubiquitination of ARMC5, suggesting that ARMC5 is a substrate for a CUL3-based ubiquitin complex. These data show a new mechanism of regulation of the ARMC5 protein and open new perspectives for understanding the pathophysiology of PBMAH. 81. Hu Y, Lao L, Mao J, Jin W, Luo H, Charpentier T, et al.: Armc5 * deletion causes developmental defects and compromises Tcell immune responses. Nat Commun 2017, 8:13834. Animal model study using Armc5 knockout mice, indicated that ARMC5 is crucial in fetal development, T-cell function and adrenal gland growth homeostasis, and that the functions of ARMC5 probably depend on interaction with multiple signaling pathways.
Current Opinion in Endocrine and Metabolic Research 2019, 8:112–121