Human Adrenal Cortex Research Articles

The Functional Glomerulosa Cells Define the Unicity of the Adult Human Adrenal Cortex

Abstract The zonation of the human adrenal cortex has long been established morphologically and histologically as three distinct layers of cells. The outer zona glomerulosa (ZG) comprises densely packed cells arranged in clusters that produce aldosterone; the zona fasciculata (ZF) is composed of cells with large cytoplasm, containing lipid droplets arranged in radial columns that synthetize cortisol; and the zona reticularis is composed of compact and pigmented cells producing androgens. The main purpose of this work was to study the expression of aldosterone synthase (CYP11B2 which catalyzes the last steps of aldosterone synthesis) and 11β-hydroxylase (CYP11B1 which catalyzes the last step of cortisol synthesis) in normal adrenal glands to address issues regarding the zonation and the fate of the cells constitutive of each zone through the expression of Ki-67 and cleaved Caspase-3. Thirty eight normal human adrenals (16 females, 22 males, ranging in age from 22 to 81 years old with a median age of 52 years old) were obtained from brain-dead organ donors (kindly provided by the Organ Transplant Clinics, University Hospital of Rouen). As early as 22 years old, we found that the histological ZG (h-ZG) does not correspond to the functional ZG (f-ZG) expressing CYP11B2. Moreover, the h-ZG CYP11B2- cells were CYP11B1+ showing that these cells ascribed to the h-ZG are in fact cortisol producing cells. The progressive replacement of CYP11B2+ cells by CYP11B1+ cells in the h-ZG might demonstrate the role of the extracellular matrix in the morphological maintenance of the adrenal cortex. Our analysis also showed that steroidogenic cells were either CYP11B1 or CYP11B2 positive. By immunofluorescence, we observed in many cases isolated or clusters of CYP11B2+ cells located deeply in the h-ZF and sometimes in the vicinity of the central vein. We were able to show that those cells were probably issued from CYP11B2+ cell clusters located in h-ZG which migrated centripetally. Ki-67 immunoreactivity was highly variable and observed throughout the entire cortex. We also found a positive correlation between the steroidogenic and endothelial cells proliferation. It is interesting to note that some Ki-67+ cells located in the h-ZG were CYP11B1+. Cortical cells positive for cleaved Caspase-3 were extremely rare but detected in all zones when present. These findings challenge the classic view of lineage conversion of differentiated ZG cells and show a new pathway where the CYP11B2+ cells migrate without changing their phenotype.

Functional Characteristic and Significance of Aldosterone-Producing Cell Clusters in Primary Aldosteronism and Age-Related Hypertension.

Primary aldosteronism (PA) is one of the most frequent curable forms of secondary hypertension. It can be caused by the overproduction of aldosterone in one or both adrenal glands. The most common subtypes of PA are unilateral aldosterone over-production due to aldosterone-producing adenomas (APA) or bilateral aldosterone over-production due to bilateral hyperaldosteronism (BHA). Utilizing the immunohistochemical (IHC) detection of aldosterone synthase (CYP11B2) has allowed the identification of aldosterone-producing cell clusters (APCCs) with unique focal localization positive for CYP11B2 expression in the subcapsular portion of the human adult adrenal cortex. The presence of CYP11B2 supports that synthesis of aldosterone can occur in these cell clusters and therefore might contribute to hyperaldosteronism. However, the significance of the steroidogenic properties of APCCs especially in regards to PA remains unclear. Herein, we review the available evidence on the presence of APCCs in normal adrenals and adrenal tissues adjacent to APAs, their aldosterone-stimulating somatic gene mutations, and their accumulation during the ageing process; raising the possibility that APCCs may play a role in the development of PA and age-related hypertension.

Aldosterone and cortisol synthesis regulation by angiotensin-(1-7) and angiotensin-converting enzyme 2 in the human adrenal cortex.

The branch of the renin--angiotensin system constituting angiotensin-(1-7) [Ang-(1-7)], the Ang II type 2 receptor, the Mas receptors and the Ang-(1-7)-forming enzyme ACE-2, by counteracting the Ang II type 1 receptor (AT1R)-mediated effects, are held to be cardiovascular protective in several conditions. However, whether Ang-(1-7) and ACE-2 are detectable in human adrenocortical tissues and whether they affect aldosterone and cortisol biosynthesis was unknown. We measured angiotensin peptides with liquid chromatography tandem-mass spectrometry and ACE-2 mRNA with digital droplet (dd)PCR in human aldosterone-producing adenoma (APA) and APA-adjacent tissue obtained from patients with primary aldosteronism. We also investigated the effects of Ang-(1-7) and the ACE-2 activator diminazene aceturate (DIZE) on aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) gene expression, in the absence or presence of the AT1R antagonist irbesartan, or of the MasR antagonist A779. APA and APA-adjacent adrenocortical tissues express ACE-2 mRNA and contain detectable amounts of Ang II and Ang-(2-8), but not of Ang I, Ang-(1-5), Ang (3-8) and Ang-(1-7). Under unstimulated and Ang II- stimulated conditions Ang-(1-7) did not blunt CYP11B1 and CYP11B2 mRNA. At supraphysiological concentrations (10-4 mol/l), Ang-(1-7) stimulated both CYP11B1 and CYP11B2 mRNA via the AT1R. The ACE-2 activator DIZE increased by 1.5-fold ACE-2 mRNA but did not blunt Ang II- upregulated CYP11B1 and CYP11B2 expression. These results do not support the hypothesis that the ACE-2/Ang-(1-7)/MasR axis play a protective role by counteracting enhanced aldosterone secretion in humans.

Adrenal cortex development and related disorders leading to adrenal insufficiency

The adult human adrenal cortex produces steroid hormones that are crucial for life, supporting immune response, glucose homeostasis, salt balance and sexual maturation. It consists of three histologically distinct and functionally specialized zones. The fetal adrenal forms from mesodermal material and produces predominantly adrenal C19 steroids from its fetal zone, which involutes after birth. Transition to the adult cortex occurs immediately after birth for the formation of the zona glomerulosa and fasciculata for aldosterone and cortisol production and continues through infancy until the zona reticularis for adrenal androgen production is formed with adrenarche. The development of this indispensable organ is complex and not fully understood. This article gives an overview of recent knowledge gained of adrenal biology from two perspectives: one, from basic science studying adrenal development, zonation and homeostasis; and two, from adrenal disorders identified in persons manifesting with various isolated or syndromic forms of primary adrenal insufficiency.

Gender differences in human adrenal cortex and its disorders

The adrenal cortex plays pivotal roles in the maintenance of blood volume, responsiveness to stress and the development of gender characteristics. Gender differences of human adrenal cortex have been recently reported and attracted increasing interests. Gender differences occur from the developing stage of the adrenal, in which female subjects had more activated stem cells with higher renewal capacity resulting in gender-associated divergent structures and functions of cortical zonations of human adrenal. Female subjects generally have the lower blood pressure with the lower renin levels and ACE activities than male subjects. In addition, HPA axis was more activated in female than male, which could possibly contribute to gender differences in coping with various stressful events in our life. Of particular interest, estrogens were reported to suppress RAAS but activate HPA axis, whereas androgens had opposite effects. In addition, adrenocortical disorders in general occur more frequently in female with more pronounced adrenocortical hormonal abnormalities possibly due to their more activated WNT and PRK signaling pathways with more abundant activated adrenocortical stem cells present in female adrenal glands. Therefore, it has become pivotal to clarify the gender influence on both clinical and biological features of adrenocortical disorders. We herein reviewed recent advances in these fields.

Primary Cultures and Cell Lines for In Vitro Modeling of the Human Adrenal Cortex.

The human adrenal cortex is a complex endocrine organ that produces mineralocorticoids, glucocorticoids and androgens. These steroids are produced in distinct cell types located within the glomerulosa, fasciculata and reticularis of the adrenal cortex. Abnormal adrenal steroidogenesis leads to a variety of diseases that can cause hypertension, metabolic syndrome, infertility and premature adrenarche. The adrenal cortex can also develop steroid-producing adenomas and rarely adrenocortical carcinomas. In vitro cell culture models provide important tools to study molecular and cellular mechanisms controlling both the physiologic and pathologic conditions of the adrenal cortex. In addition, the presence of multiple steroid-metabolizing enzymes within adrenal cells makes it a model for defining possible endocrine disruptors that might block these enzymes. The regulation and dysregulation of human adrenal steroid production and cell division/tumor growth can be studied using freshly isolated cells but this requires access to human adrenal glands, which are not available to most investigators. Immortalized human adrenocortical cell lines have proven to be of considerable value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis and tumorigenesis. Current human adrenal cell lines include the original NCI-H295 and its substrains: H295A, H295R, HAC13, HAC15, HAC50 and H295RA as well as the recently established MUC-1, CU-ACC1 and CU-ACC2. The current review will discuss the use of primary cultures of fetal and adult adrenal cells as well as adrenocortical cell lines as in vitro models for the study of human adrenal physiology and pathophysiology.

Characterization of Group I Metabotropic Glutamate Receptors in Rat and Human Adrenal Glands.

Glutamate and its receptors have been demonstrated to promote both basal and nicotine-evoked catecholamine release in bovine chromaffin cells. Multiple glutamate receptors, including metabotropic glutamate receptors (mGluRs), are found in the adrenal glands of several species, as well as in chromaffin cells. However, there is limited information available regarding the expression of glutamate metabotropic receptor (GRM)1-8 mRNAs and the detailed localization of group I mGluRs (mGluR1 and mGluR5) in the rat and human adrenal cortex and medulla. Therefore, we examined mRNA expression of GRM1-8 subunits using reverse transcription-polymerase chain reaction (RT-PCR) and the distribution of mGluR1 and mGluR5 by immunostaining. The results showed that the GRM1-8 mRNAs were expressed in both the cortex and medulla of rat and human adrenal glands with the exception of GRM1, which was not detectable in the rat adrenal cortex. Immunostaining of mGluR1 revealed that it was localized only in the adrenal medulla of rats but was present in both the adrenal cortex and medulla in humans. In the adrenal medulla, the central part of the adrenal glands, mGluR1 was detected in chromaffin cells but not in nerve fibers and ganglion cells. Immunoactivity of mGluR5 was visible in the capillary wall throughout the adrenal cortex and medulla in rat and human samples. Its immunoactivity was also observed in ganglion cells in the rat adrenal medulla. There was no mGluR5 immunoactivity detected in chromaffin cells and nerve fibers in the rat and human adrenal medulla. Using dissected rat adrenal medulla as a model, we found that treatment with a mGluR1 agonist activated extracellular signal-regulated kinase (ERK) 1/2 and increased the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis. Moreover, these results showed that mGluR1 signaling was involved in hypoxia-induced upregulation of TH in the rat adrenal medulla. This study shows the expression of GRM1-8 mRNAs in rat and human adrenal glands and indicates that glutamate, through the activation of mGluRs, may play various physiological roles in the adrenal gland. Furthermore, mGluR1 may be involved in catecholamine biosynthesis by regulating TH, and mGluR5 may affect cortical and medullar hormone levels by regulating microvascular function.

SUN-210 Differentially Expressed Mirnas in Zona Reticularis (ZR) Cells of the Human Adrenal Cortex

The human adrenal cortex, involved in adaptive responses to stress, fluid homeostasis, and secondary sexual characteristics, arises from a tightly regulated development of a zone and cell type-specific secretory pattern. However, the molecular mechanisms governing adrenal zonation, particularly postnatal ZR development, which produces adrenal androgens in a life-time-specific manner, remain poorly understood. The hallmark of ZR is the low expression of type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). However, the mechanisms underlying HSD3B2 downregulation in the ZR remain unknown. MiRNAs are seen as regulators of cell phenotypes. The objective of the study was to compare miRNA expression profiles in human adrenal ZR and zona fasciculata (ZF). ZF and ZR were microdissected from human adrenals tissues (n=5, from boys aged 16, 17 and 19 yr and girls aged 12 and 16 yr) by laser capture microdissection (LCM). Total RNA was extracted from 5 ZF/ZR pairs and next-generation sequencing (NGS) was used to perform the microRNA expression profiling. 281 mature microRNAs were identified in human adrenal cortex. Among them, 7 microRNAs were significantly differentially expressed between ZF and ZR. The expression of miR-375-3p, miR-483-3p and miR-7-5p was higher in ZR compared with its paired ZF by 2-fold or greater. Multiple available bioinformatic algorithms (TargetScan, miRanda, DianaLab and PicTar) were employed to search for its target genes. Among predicted target genes, several genes (GATA-6, GATA-4, SF1, NR4A2, and IGF-1) are known to be involved in HSD3B2 regulation. In summary, LCM combined with NGS provided a robust approach to explore the adrenal zone-specific micro-RNA profiling. Our data gave first hints that miRNAs might be novel regulatory modules associated with human adrenal ZR cell-specific transcript regulation underlying developmental androgen production.

OR03-03 New Insights into the Functional Human Adrenal Cortex Zonation

The zonation of the human adrenal cortex has long been established morphologically and histologically as three distinct layers of cells. The outer zona glomerulosa (ZG) comprises densely packed cells arranged in clusters that produce aldosterone; the zona fasciculata (ZF) is composed of cells with large cytoplasm, containing lipid droplets arranged in radial columns that synthetize cortisol; and the zona reticularis is composed of compact and pigmented cells producing androgens. The main purpose of this work was to study the expression of aldosterone synthase (CYP11B2 which catalyzes the last steps of aldosterone synthesis) and 11β-hydroxylase (CYP11B1 which catalyzes the last step of cortisol synthesis) in normal adrenal glands to address issues regarding the zonation and the fate of the cells constitutive of each zone through the expression of Ki-67 and cleaved Caspase-3. Thirty eight normal human adrenals (16 females, 22 males, ranging in age from 22 to 81 years old with a median age of 52 years old) were obtained from brain-dead organ donors (kindly provided by the Organ Transplant Clinics, University Hospital of Rouen). As early as 22 years old, we found that the histological ZG (h-ZG) does not correspond to the functional ZG (f-ZG) expressing CYP11B2. Moreover, the h-ZG CYP11B2- cells were CYP11B1+ showing that these cells ascribed to the h-ZG are in fact cortisol producing cells. The progressive replacement of CYP11B2+ cells by CYP11B1+ cells in the h-ZG might demonstrate the role of the extracellular matrix in the morphological maintenance of the adrenal cortex. Our analysis also showed that steroidogenic cells were either CYP11B1 or CYP11B2 positive. By immunofluorescence, we observed in many cases isolated or clusters of CYP11B2+ cells located deeply in the h-ZF and sometimes in the vicinity of the central vein. We were able to show that those cells were probably issued from CYP11B2+ cell clusters located in h-ZG which migrated centripetally. Ki-67 immunoreactivity was highly variable and observed throughout the entire cortex. We also found a positive correlation between the steroidogenic and endothelial cells proliferation. It is interesting to note that some Ki-67+ cells located in the h-ZG were CYP11B1+. Cortical cells positive for cleaved Caspase-3 were extremely rare but detected in all zones when present. These findings challenge the classic view of lineage conversion of differentiated ZG cells and show a new pathway where the CYP11B2+ cells migrate without changing their phenotype.

SUN-LB43 DLK1 Expressing Cells Contribute to the Zonation of the Adrenal Gland

The adrenal cortex is a dynamic organ that undergoes self-renewal and remodeling in response to the demand for steroids. In the mouse it is divided into two concentric layers, the outer zona glomerulosa and the inner zona fasciculata (ZF), that secrete aldosterone and corticosterone, respectively. Cell fate mapping studies have shown that the maintenance of the cortex relies on a pool of stem/progenitor cells located in the capsular and subcapsular compartments. Two interconnected cell populations have been identified, subcapsular undifferentiated cells secreting the morphogen Sonic Hedgehog (Shh) and capsular Gli1+ cells, which can transduce the Shh signal (1); both populations are precursors of steroidogenic cells and newly formed cells migrate in a centripetal fashion to repopulate the gland until they reach the juxtamedullary region where they undergo senescence and apoptosis. Moreover, our lab has shown that the Notch atypical ligand Delta-Like homologue 1 (Dlk1) is expressed in partially undifferentiated cells of the subcapsular region in rat (2)and human (3)adrenals, whilst it is mostly expressed in capsular cells in mice (4,5). To investigate whether Dlk1 expressing cells contribute to the zonation of the adrenal cortex we conducted lineage tracing analyses using a tamoxifen inducible Dlk1CreERT2mouse model carrying the R26tdTom reporter. Pregnant dames were injected with tamoxifen at embryonic day (e) 12.5 and pups were culled at postnatal day (p) 10 and p38. Analysis of tdTomato expression showed that 35% (p10) and 24% (p38) of Steroidogenic Factor 1(Sf1)+cortical cells were tdTomato+, revealing that capsular Dlk1+cells are steroidogenic precursors. On the other hand, postnatal tamoxifen injections (p0) showed tdTomato+/Sf1+ cells only in 1-2% in cortical cells after 24-months chase, suggesting that the contribution of Dlk1+cells to adrenocortical self-renewal is limited postnatally.However, the Dlk1+population could be reactivated in the adult mouse treated with dexamethasone and was shown to contribute to the regeneration of the ZF once dexamethasone treatment was ceased. 1. King P, et al. Shh signaling regulates adrenocortical development and identifies progenitors of steroidogenic lineages. Proc Natl Acad Sci(2009) 106:21185-211902. Guasti L, et al. Dlk1 Up-Regulates Gli1 Expression in Male Rat Adrenal Capsule Cells Through the Activation of β1 Integrin and ERK1/2. Endocrinology(2013) 154:4675-46843. Hadjidemetriou I, et al. DLK1/PREF1 marks a novel cell population in the human adrenal cortex. J Steroid Biochem Mol Biol(2019) 193:1054224. Guasti L, Candy Sze WC, McKay T, Grose R, King PJ. FGF signalling through Fgfr2 isoform IIIb regulates adrenal cortex development. Mol Cell Endocrinol(2013) 371:182-1885. Heikkilä M, et al. Wnt-4 Deficiency Alters Mouse Adrenal Cortex Function, Reducing Aldosterone Production. Endocrinology(2002) 143:4358-4365

SUN-073 Urine Steroid Profile of Girls with Premature Adrenarche

Background: Adrenarche describes the development of the human adrenal cortex when the zona reticularis increases the synthesis of C19 steroids (DHEA/-S). Girls with premature adrenarche have a higher risk to develop adverse outcomes including polycystic ovary syndrome and metabolic syndrome later in life. The role of novel biosynthetic pathways of androgen production in health and disease remains largely unsolved. Objective: This study aimed to compare the urinary steroid metabolome between girls with premature adrenarche and healthy girls with focus on metabolites originating of novel, alternate androgen pathways. Methods: In 23 girls with premature adrenarche (median age 7 years) and 22 healthy, age-matched girls, we measured 39 steroid metabolites comprising progesterones, corticosterones, aldosterone, androgens, estrogens and glucocorticoids in the urine collected over 24 h by gas chromatography mass spectrometry. We compared metabolites and metabolite ratios between both groups of girls using Mann-Whitney tests with Bonferroni correction to account for multiple testing. Results: Girls with premature adrenarche were heavier than healthy girls (median weight 26.2 kg vs. 21.5 kg, p=0.003) and had a higher BMI SDS (0.8 vs -0.3, p=0.013). Gestational age and birth weight was similar between groups. Overall androgen excretion was different between groups, in particular amounts of androsterone, etiocholanolone, androstanediol, dehydroepiandrosterone, androstenediol, androstenetriol and pregnenetriol were higher in girls with premature adrenarche than in healthy girls (p<0.05). Some of these metabolites originate from alternate androgen pathways, e.g. androsterone. We found no differences in progesterones, corticosterones, aldosterone, estrogens and glucocorticoids, except for 20β-dihydrocortisone, which was higher in girls with premature adrenarche. Activities of 17βHSD and of 17,20-lyase via the Δ4pathway were higher in girls with premature adrenarche than in healthy girls. Conclusions: Girls with premature adrenarche produce more androgens than healthy girls of similar age. The urinary steroid signature of adrenarche includes metabolites of alternate pathways. Androstanediol seems a marker of adrenarche. Future studies should assess whether the steroid signature of adrenarche is just appearing earlier in girls with premature adrenarche or earlier and different compared to adrenarche at normal timing.

SAT-173 SAMD9 (Sterile Alpha Motif Domain-Containing 9) Expression in Adrenocortical Tumors

Introduction: SAMD9 variants are associated with colon, breast and lung tumors. SAMD9 mutations result in adrenal hypoplasia, suggesting its importance in adrenal development. We investigated the contribution of abnormal expression of SAMD9 and its homologous, SAMD9L, to the pathogenesis of pediatric adrenocortical tumors (pACT) Objective: To evaluate the involvement of SAMD9 and SAMD9L in normal human adrenal cortex development and adrenocortical tumorigenesis, as well as to evaluate their association with tumor presentation and patient outcome. Methods: pACT samples (n= 72), normal pediatric adrenal cortices (n= 11), and normal fetal and post-natal adrenals (20 weeks of gestation to 10 years of age; n= 51) were enrolled. Protein expression of SAMD9 (immunohistochemistry) and SAMD9/SAMD9L mRNA levels were evaluated (qPCR). The associations between SAMD9/SAMD9L expression in pACT with tumor presentation (P53 p.R337H genotype and metastasis occurrence) and patient outcome (Overall (OS) and Disease-Free Survival) were analyzed. In silico, publicly available data from pediatric patients with ACT available in the Gene Expression Omnibus were used to evaluate the aforementioned associations with SAMD9 (GSE76021) and SAMD9L (GSE76019) mRNA levels. In vitro, SAMD9 subcellular localization pattern was investigated in the ACT cell line NCI-H295R (immunofluorescence). Results: Nuclear and cytoplasmic SAMD9 expression was observed throughout all different phases of adrenal development evaluated. However, in pACT samples, 26% presented nuclear and 86% cytoplasmic immunostaining. In line, NCI-H295R cells presented mostly cytoplasmic SAMD9 immunostaining under basal conditions. No differential expression was observed between SAMD9 or SAMD9L mRNA levels in pACT and in normal pediatric adrenals. Moreover, no association between SAMD9 immunostaining, SAMD9 or SAMD9L mRNA levels, tumor presentation and patient outcome were observed in our cohort, nor in the in silico evaluation. Conclusion: SAMD9 is nuclear and cytoplasmic expressed during adrenal cortex adrenal development and its loss of function is associated with adrenal hypoplasia. On the other hand, SAMD9 is mostly cytoplasmic expressed in pACT and immortalized NCI-H295R cells. No differential expression of SAMD9 nor its counterpart SAMD9L mRNA were associated with tumor presentation and pediatric patient outcome.

Angiotensin II-upregulated MAP kinase phosphatase-3 modulates FOXO1 and p21 in adrenocortical H295R cells

MAPK phosphatases (MKP) downregulate the activity of mitogen-activated protein kinases (MAPK), such as ERK1/2, and modulate the processes regulated by these kinases. ERK1/2 participate in a wide range of processes including tissue-specific hormone-stimulated steroidogenesis. H295R cells are a suitable model for the study of human adrenal cortex functions, particularly steroid synthesis, and respond to angiotensin II (Ang II) triggering ERK1/2 phosphorylation in a transient fashion. MKP-3 dephosphorylates ERK1/2 and, as recently reported, forkhead box protein 1 (FOXO1). Here, we analyzed MKP-3 expression in H295R cells and its putative regulation by Ang II. Results showed the expression of MKP-3 full length (L) and a short splice variant (S), and the upregulation of both isoforms by Ang II. L and S messenger and protein levels increased 30 min after Ang II stimulation and declined over the next 3 h, a temporal frame compatible with ERK1/2 dephosphorylation. In addition, FOXO1 activation is known to include its dephosphorylation and nuclear translocation. Therefore, we analyzed the effect of Ang II on FOXO1 modulation. Ang II induced FOXO1 transient phosphorylation and translocation and also the induction of p21, a FOXO1-dependent gene, whereas MKP-3 knock-down reduced both FOXO1 translocation and p21 induction. These data suggest that, through MKP-3, Ang II counteracts its own effects on ERK1/2 activity and also triggers the activation of FOXO-1 and the induction of cell cycle inhibitor p21. Taken together, the current findings reveal the participation of MKP-3 not only in turn–off but also in turn-on signals which control important cellular processes.

Cellular and Functional Defects in Aldosteronism-linked Cytosolic Domain Mutations in GIRK4 (KCNJ5)

GIRK4 (KCNJ5) is one of four subunits of the G-protein gated inwardly rectifying potassium channels (GIRK1-4). GIRK4 subunits are abundant in heart and adrenal cortex, and form homo- or heterotetramers with GIRK1. GIRK channels mediate inhibitory transmission via Gi/o-protein coupled receptors, with Gβγ subunits as their main gating factor. KCNJ5 mutations were causally linked to primary aldosteronism (PA). The leading concept for PA-related mutations in KCNJ5 is that mutations in the pore region cause loss of K+ selectivity, which increases Na+ conductance and membrane depolarization followed by Ca2+ influx and aldosterone secretion. This mechanism later was extended to mutations in cytosolic N- and C-terminal domains far from the selectivity filter: GIRK4R52H, GIRK4E246K, and GIRK4G247R. Previously, using Xenopus laevis oocytes, we have shown that the activity of heterotetrameric GIRK1/4R52H and GIRK1/4E246K channels was reduced. We also showed that the mutations did not affect the selectivity or inward rectification. Here, we show that the activity of GIRK1/4G247R was not different from GIRK1/4WT. We corroborated these results in human adrenal cortex cell line (HAC15). Confocal imaging revealed that abundance in plasma membrane of mutated homo- or heterotetrameric channels was significantly reduced. Moreover, mutated homotetrameric channels were non-functional; however, VU0529331, an opener of GIRK4 homotetramers, rescued the activity of GIRK4G247R. Using Fluorescence Resonance Energy Transfer (FRET), we demonstrate that interaction of Gβγ with GIRK1/4R52H and GIRK1/4E246K is significantly impaired in a wide range of acceptor/donor molar ratios. Interaction with GIRK1/4G247R was only slightly impaired. Our results suggest an impairment of both Gβγ interaction and expression for GIRK4R52H and GIRK4E246K, but mainly a reduction in expression for homotetrameric (and not heterotetrameric) GIRK4G247R. Furthermore, these results rule out a previously proposed mechanism for R52H and E246K mutants, and indicate potential ways of clinical treatment.

Дослідження механізмів цитотоксичної дії адренокортиколітичних та антимітотичних препаратів в адренокортикоцитах людини

Антимітотичні агенти наразі широко використовуються як протипухлинні терапевтичні засоби. Проте їх використання має деякі обмеження, оскільки останні є субстратом Р-глікопротеїну, який визначає резистентність клітин до протипухлинних препаратів. Мета — дослідити вплив селективного інгібітору Р-глікопротеїну PGP4008 на реалізацію ефекту адренокортиколітичної сполуки орто, пара’-дихлордифенілдихлоретану (о, п’-ДДД) та антимітотичних сполук колхіцину й цитохалазину В в адренокортикоцитах людини. Матеріал і методи. Дослідження in vitro проводили на диспергованих клітинах, отриманих із позапухлинної тканини кори надниркових залоз людини. До середовища інкубації додавали розчин о, п’-ДДД (кінцеві концентрації 10 мкмоль/л і 50 мкмоль/л), колхіцин (10 мкмоль/л) і цитохалазин В (10 мкмоль/л). Селективний інгібітор Р-глікопротеїну PGP4008 вносили до інкубаційного середовища в кінцевій концентрації 50 мкмоль/л. Інкубація тривала 18 год за 37 °C. Оцінку цитотоксичної дії досліджуваних сполук проводили за підрахунком кількості загиблих клітин, забарвлених 0,4% трипановим синім у камері Горяєва. Результати. Передінкубація адренокортикальних клітин з о, п’-ДДД, колхіцином і цитохалазином В призводить до збільшення відсотка мертвих клітин. Додавання до живильного середовища PGP4008 посилювало цитотоксичний ефект досліджуваних сполук. Висновок. Проведені дослідження показали, що використання о, п’-ДДД, колхіцину та цитохалазину В з інгібітором Р-глікопротеїну PGP4008 є перспективним для подальшого передклінічного вивчення.

Splice-variant-specific effects of primary aldosteronism point mutations on human CaV3.2 calcium channels

CaV3.2 calcium channels play important roles in both neural excitability and aldosterone secretion. Recent clinical studies found four germline mutations (S196 L, M1549I, V1951E and P2083 L) in CaV3.2 channels. All four mutations caused primary aldosteronism (PA), while only the M1549I mutation resulted in obvious neural malfunctions besides PA. In human, there are two major CaV3.2 channel gene (CACNA1H) splice variants, either with or without exon 26. In this study, we tested the expression of the two CACNA1H splice variants in zona glomerulosa (ZG) cells of human adrenal cortex and the possibility that CaV3.2 (-26) and CaV3.2 (+26) channels have different functional responses to the four PA mutations. We found that human ZG cells only express long form CaV3.2(+26) channels. The M1549I mutation slowed the inactivation of CaV3.2(+26) more than 5 fold, and CaV3.2(-26) more than 2 fold. The S196 L, V1951E and P2083 L mutations accelerated channel recovery from inactivation for CaV3.2(+26), but not CaV3.2(-26) channels. All four mutations resulted in gain of function of CaV3.2(+26) channels, leading to overproduction of aldosterone. In conclusion, the four PA mutations caused more profound changes on CaV3.2 (+26) currents than on CaV3.2 (-26) currents, and except the M1549I mutation, the S196 L, V1951E and P2083 L have little effect on the electrophysiological properties of CaV3.2(-26) currents, which may partially explain the limitation of the phenotype associated with the V1951E, S196 L and P2083 L germline mutations to PA.

Correlation Between Telomere Attrition of Zona Fasciculata and Adrenal Weight Reduction in Older Men.

Although numerous theories are reported on sex differences in longevity, the underlying biological mechanisms remain unknown. We previously reported that telomere length in the zona reticularis cells of the human adrenal cortex was significantly longer in older than that in younger subjects. However, we could not evaluate sex differences in the telomere lengths. To compare the telomere lengths of adrenocortical and adrenal medullar cells between men and women from infancy through older adulthood. Adrenal glands of 30 male (aged 0 to 100 years) and 25 female (aged 0 to 104 years) autopsied subjects were retrieved from autopsy files. Using quantitative fluorescence in situ hybridization, relative telomere lengths were determined in the parenchymal cells of the 3 adrenocortical zones and medulla. Age-related changes in the weight of adrenal glands were also investigated. Older male subjects (aged 65 years or older) had significantly shorter telomere lengths in zona fasciculata (ZF) cells compared to the corresponding female subjects. In men, older subjects exhibited a significant age-related reduction in adrenal weight; however, no age-related changes in adrenal weight were detected in women. Telomere attrition of ZF cells was correlated with adrenal weight reduction in older men but not in older women, suggesting a decreased number of ZF cells in older men. This may help us understand the possible biological mechanisms of sex difference in longevity of humans.

DLK1/PREF1 marks a novel cell population in the human adrenal cortex

The adrenal cortex governs fundamental metabolic processes though synthesis of glucocorticoid, mineralocorticoids and androgens. Studies in rodents have demonstrated that the cortex undergoes a self-renewal process and that capsular/subcapsular stem/progenitor cell pools differentiate towards functional steroidogenic cells supporting the dynamic centripetal streaming of adrenocortical cells throughout life. We previously demonstrated that the Notch atypical ligand Delta-like homologue 1 (DLK1)/preadipocyte factor 1 (PREF1) is expressed in subcapsular Sf1 and Shh-positive, CYP11B1-negative and CYP11B2-partially positive cortical progenitor cells in rat adrenals, and that secreted DLK1 can modulate GLI1 expression in H295R cells. Here we show that the human adrenal cortex remodels with age to generate clusters of relatively undifferentiated cells expressing DLK1. These clusters (named DLK1-expressing cell clusters or DCCs) increased with age in size and were found to be different entities to aldosterone-producing cell clusters, another well-characterized and age-dependent cluster structure. DLK1 was markedly overexpressed in adrenocortical carcinomas but not in aldosterone-producing adenomas. Thus, this data identifies a novel cell population in the human adrenal cortex and might suggest a yet-to be identified role of DLK1 in the pathogenesis of adrenocortical carcinoma in humans.

Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrançois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

Reappraisal of the human adrenal cortex and fate

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)