Regulation of Corticosteroidogenic Genes by MicroRNAs

The loss of normal regulation of corticosteroid secretion is important in the development of cardiovascular disease. We previously showed that microRNAs regulate the terminal stages of corticosteroid biosynthesis. Here, we assess microRNA regulation across the whole corticosteroid pathway. Knockdown of microRNA using Dicer1 siRNA in H295R adrenocortical cells increased levels of CYP11A1, CYP21A1, and CYP17A1 mRNA and the secretion of cortisol, corticosterone, 11-deoxycorticosterone, 18-hydroxycorticosterone, and aldosterone. Bioinformatic analysis of genes involved in corticosteroid biosynthesis or metabolism identified many putative microRNA-binding sites, and some were selected for further study. Manipulation of individual microRNA levels demonstrated a direct effect of miR-125a-5p and miR-125b-5p on CYP11B2 and of miR-320a-3p levels on CYP11A1 and CYP17A1 mRNA. Finally, comparison of microRNA expression profiles from human aldosterone-producing adenoma and normal adrenal tissue showed levels of various microRNAs, including miR-125a-5p to be significantly different. This study demonstrates that corticosteroidogenesis is regulated at multiple points by several microRNAs and that certain of these microRNAs are differentially expressed in tumorous adrenal tissue, which may contribute to dysregulation of corticosteroid secretion. These findings provide new insights into the regulation of corticosteroid production and have implications for understanding the pathology of disease states where abnormal hormone secretion is a feature.

Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands

Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na+/K+transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA.

Differential expression of microRNA-675, microRNA-139-3p and microRNA-335 in benign and malignant adrenocortical tumours

BackgroundFor the clinical management of adrenocortical neoplasms it is crucial to correctly distinguish between benign and malignant tumours. Even histomorphologically based scoring systems do not allow precise separation in single lesions, thus novel parameters are desired which offer a more accurate differentiation. The tremendous potential of microRNAs (miRNAs) as diagnostic biomarkers in surgical pathology has recently been shown in a broad variety of tumours.MethodsIn order to elucidate the diagnostic impact of miRNA expression in adrenocortical neoplasms, a cohort of 20 adrenocortical specimens including normal adrenal tissue (n=4), adrenocortical adenomas (ACAs) (n=9), adrenocortical carcinomas (ACCs) (n=4) and metastases (n=3) was analysed using TaqMan low density arrays to identify specific miRNA profiles in order to distinguish between benign and malignant adrenocortical lesions. Results were validated in a validation cohort (n=16).ResultsConcerning the differential diagnosis of ACAs and ACCs, 159 out of 667 miRNAs were up- and 89 were down-regulated in ACAs. Using real-time PCR analysis of three of the most significantly expressed single key miRNAs allowed separation of ACAs from ACCs. ACCs exhibited significantly lower levels of miR-139-3p (up to 8.49-fold, p<0.001), miR-675 (up to 23.25-fold, p<0.001) and miR-335 (up to 5.25-fold, p<0.001). A validation cohort of 16 specimen with known Weiss score showed up-regulation of miR-335 and miR-675 in the majority of cases with probable malignant course, although overlapping values exist.ConclusionmiRNA profiling of miR-675 and miR-335 helps in discriminating ACCs from ACAs. miRNA analysis may indicate malignant behaviour in cases with indeterminate malignant potential.

Major hyperestrogenism in a feminizing adrenocortical adenoma despite a moderate overexpression of the aromatase enzyme

A 30-year-old male was referred for the rapid development of gynecomastia, and dramatic hyperestrogenemia was assessed: plasma estrone, estradiol but also cortisol were not suppressed by high-dose dexamethasone, while gonadotropin pulsatility was completely abolished. A 60-mm right adrenal tumor was evidenced on computed tomography-scan, and the patient underwent adrenalectomy. The tumor was found to express a moderate increase in aromatase activity compared with adjacent non-neoplastic adrenal tissue. Quantitative RT-PCR also showed a weak and non-significant increase in total aromatase mRNA in the tumor compared with normal adrenal tissue. Aromatase transcripts were mainly promoter PII-derived, but different patterns of aromatase minor transcripts were found: promoter I.3- and I.6-derived transcripts were identified in the tumor, while only promoter I.4-derived transcripts were found in normal adrenal. This case report demonstrates that a sharp aromatase overexpression is not a prerequisite for clinical and biochemical hyperestrogenism, and further characterizes the aromatase promoter utilization in this feminizing adrenocortical tumor and in the normal adrenal cortex.

IN VITRO RESPONSES OF FOCAL HYPERPLASTIC TISSUE OF THE HUMAN ADRENAL ZONA FASCICULATA TO ACTH

ABSTRACT The temporal cAMP, cortisol and aldosterone responses to ACTH of focal hyperplasia of the zona fasciculata and of normal human adrenocortical tissue were investigated. ACTH significantly increased cAMP levels (1 min) and cortisol output (2 min) in normal adrenal tissue but not in hyperplastic tissue. However, following ACTH treatment cortisol and aldosterone production were depressed in the abnormal adrenal tissue below the untreated or the ACTH stimulated normal adrenal tissue. In addition, basal cortisol and aldosterone production of the hyperplastic adrenal tissue was elevated above that of the normal adrenal tissue. These findings suggest that the cAMP second messenger concept may be only one of several mechanisms in the modulation of human adrenocortical function.

ZUR FRAGE DER BIOSYNTHESE VON ALDOSTERON UND 11-DESOXYCORTICOSTERON IN DER NEBENNIERE UND IM ADENOM BEI EINEM PATIENTEN MIT PRIMÄREM ALDOSTERONISMUS

ABSTRACT In a 19-year old male patient, suffering from primary aldosteronism, the biosynthesis of 11-deoxycorticosterone and aldosterone was studied in slices of normal and adenomatous adrenal tissue; [4-14C] progesterone and [4-14C] 17α-hydroxyprogesterone were used as substrates. In addition, the urinary excretion of 1 1-deoxycorticosterone, tetrahydro-11-deoxycorticosterone and aldosterone was determined before and after operation. As compared with normal adrenal tissue, a very high activity of the 21-hydroxylase towards progesterone (metabolite: 11-deoxycorticosterone) as well as 17α-hydroxyprogesterone (metabolite: 17α-hydroxy-11-deoxycorticosterone) was found in the adenoma. In contrast, the activity of the 11β-hydroxylase was much less in the adenoma than in the normal adrenal tissue. In the absence of cofactors, only traces of aldosterone were detected in the experiments with slices of the adenoma, whereas a normal rate of production was observed in the experiments with the adrenal slices. The excretion of aldosterone in urine varied between 29.4 and 70.7 μg/24 h before operation; it was unaffected by dietetic measures, thus indicating an autonomy of the tumour. After operation, the concentration of aldosterone in urine fell to normal values (6.1–9.1 μg/24 h). The excretion of free 11-deoxycorticosterone (0.2–2.0 μg/24 h) and of its tetrahydroderivative (31.7–40.4 μg/24 h) was in the normal range before as well as after operation. The increased formation of 11-deoxycorticosterone and the decreased formation of aldosterone in the adenoma under in vitro conditions stand in contrast to the normal excretion of 11-deoxycorticosterone and the increased excretion of aldosterone in urine before operation. This discrepancy may be explained by the deficiency of cofactors in the slices of the adenoma. The results obtained support the view that, in steroid producing organs with high activities, only limited conclusions can be drawn from in vitro experiments to the situation in vivo.