Somatic mutations have been identified in more than half of aldosterone-producing adenomas (APAs) through mutation hotspot sequencing. The underlying pathogenesis of inappropriate aldosterone synthesis in the remaining population is still unknown.

Aldosterone synthase is the key rate-limiting enzyme in adrenal aldosterone production, and induction of its gene (CYP11B2) results in the progression of hypertension. As hypertension is a frequent complication among patients with diabetes, we set out to elucidate the link between diabetes mellitus and hypertension. We examined the effects of high glucose on CYP11B2 expression and aldosterone production using human adrenal H295R cells and a stable H295R cell line expressing a CYP11B2 5'-flanking region/luciferase cDNA chimeric construct. d-glucose (d-glu), but not its enantiomer l-glucose, dose dependently induced CYP11B2 transcription and mRNA expression. A high concentration (450 mg·dL-1) of d-glu time dependently induced CYP11B2 transcription and mRNA expression. Moreover, high glucose stimulated secretion of aldosterone into the media. Transient transfection studies using deletion mutants/nerve growth factor-induced clone B (NGFIB) response element 1 (NBRE-1) point mutant of CYP11B2 5'-flanking region revealed that the NBRE-1 element, known to be activated by transcription factors NGFIB and NURR1, was responsible for the high glucose-mediated effect. High glucose also induced the mRNA expression of these transcription factors, especially that of NURR1, but NURR1 knockdown using its siRNA did not affect high glucose-induced CYP11B2 mRNA expression. Taken together, it is speculated that high glucose may induce CYP11B2 transcription via the NBRE-1 element in its 5'-flanking region, resulting in the increase in aldosterone production although high glucose-induced NURR1 is not directly involved in the effect. Additionally, glucose metabolism and calcium channels were found to be involved in the high glucose effect. Our observations suggest one possible explanation for the high incidence of hypertension in patients with diabetes.

The emergence of the occipital alpha rhythm on brain electroencephalogram (EEG) is associated with brain activity in the cerebral neocortex and deep brain structures. To further understand the mechanisms of alpha rhythm power fluctuation, we performed simultaneous EEGs and functional magnetic resonance imaging recordings in human subjects during a resting state and explored the dynamic relationship between alpha power fluctuation and blood oxygenation level-dependent (BOLD) signals of the brain. Based on the frequency characteristics of the alpha power time series (APTS) during 20-minute EEG recordings, we divided the APTS into two components: fast fluctuation (0.04-0.167 Hz) and slow fluctuation (0-0.04 Hz). Analysis of the correlation between the MRI signal and each component revealed that the slow fluctuation component of alpha power was positively correlated with BOLD signal changes in the brain stem and the medial part of the thalamus and anterior cingulate cortex, while the fast fluctuation component was correlated with the lateral part of the thalamus and the anterior cingulate cortex, but not the brain stem. In summary, these data suggest that different subcortical structures contribute to slow and fast modulations of alpha spectra on brain EEG.

Genetic alterations in signaling pathways that control cell-cycle progression, apoptosis, and cell growth are common hallmarks of cancer, but the extent, mechanisms, and co-occurrence of alterations in these pathways differ between individual tumors and tumor types. Using mutations, copy-number changes, mRNA expression, gene fusions and DNA methylation in 9,125 tumors profiled by The Cancer Genome Atlas (TCGA), we analyzed the mechanisms and patterns of somatic alterations in ten canonical pathways: cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGFβ signaling, p53 and β-catenin/Wnt. We charted the detailed landscape of pathway alterations in 33 cancer types, stratified into 64 subtypes, and identified patterns of co-occurrence and mutual exclusivity. Eighty-nine percent of tumors had at least one driver alteration in these pathways, and 57% percent of tumors had at least one alteration potentially targetable by currently available drugs. Thirty percent of tumors had multiple targetable alterations, indicating opportunities for combination therapy.

In the adrenal gland, neuroendocrine cells that synthesize catecholamines and epithelial cells that produce steroid hormones are united beneath a common organ capsule to function as a single stress-responsive organ. The functional anatomy of the steroid hormone-producing adrenal cortex and the catecholamine-producing medulla is ill defined at the level of small molecules. Here, we report a comprehensive high-resolution mass spectrometry imaging (MSI) map of the normal human adrenal gland. A large variety of biomolecules was accessible by matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI, including nucleoside phosphates indicative of oxidative phosphorylation, sterol and steroid metabolites, intermediates of glycolysis and the tricarboxylic acid cycle, lipids, and fatty acids. Statistical clustering analyses yielded a molecularly defined adrenal anatomy of 10 distinct molecular zones including a highly structured corticomedullary interface. By incorporating pathway information, activities of carbohydrate, amino acid, and lipid metabolism as well as endocrine bioactivity were revealed to be highly spatially organized, which could be visualized as different molecularly defined zones. Together, these findings provide a molecular definition of human adult adrenal gland structure beyond classical histological anatomy.

Voltage-gated L-type calcium channel (CaV) isoforms are well known to play pivotal tissue-specific roles not only in vasoconstriction but also in adrenocortical steroidogenesis including aldosterone biosynthesis. Alpha-1C subunit calcium channel (CC) (CaV1.2) is the specific target of anti-hypertensive CC blockers (CCBs) and its Alpha-1D subunit (CaV1.3) regulates depolarization of cell membrane in aldosterone-producing cells. Direct effects of CCBs on aldosterone biosynthesis were previously postulated but their intra-adrenal distribution and effects on steroid production in primary aldosteronism (PA) patients have remained virtually unknown. In this study, frozen tissue specimens constituting tumor, adjacent adrenal gland and peri-adrenal adipose tissues of nine aldosterone-producing adenoma (APA) cases were examined for visualization of amlodipine and aldosterone themselves using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Liquid chromatography-mass spectrometry (LC-MS) analysis was also performed to quantify amlodipine and 17 adrenal steroids in those cases above and compared the findings with immunohistochemical analysis of steroidogenic enzymes and calcium channels (CaV1.2 and CaV1.3). Effects of amlodipine on mRNA level of aldosterone biosynthetic enzymes were also explored using human adrenocortical carcinoma cell line (H295R). Amlodipine-specific peak (m/z 407.1 > 318.1) was detected only in amlodipine treated cases. Accumulation of amlodipine was marked in adrenal cortex compared to peri-adrenal adipose tissues but not significantly different between APA tumors and adjacent adrenal glands, which was subsequently confirmed by LC-MS quantification. Intra-adrenal distribution of amlodipine was generally consistent with that of CCs. In addition, quantitative steroid profiles using LC-MS and in vitro study demonstrated the lower HSD3B activities in amlodipine treated cases. Immunoreactivity of CaV1.2 and HSD3B2 were also correlated. We report the first demonstration of specific visualization of amlodipine in human adrenal tissues by MALDI-MSI. Marked amlodipine accumulation in the adrenal glands suggested its direct effects on steroidogenesis in PA patients, possibly targeting on CaV1.2 and suppressing HSD3B activity.

Results of previous studies demonstrated clear racial differences in the prevalence of somatic mutations among patients with aldosterone-producing adenoma (APA). For instance, those in East Asian countries have a high prevalence of somatic mutations in KCNJ5, whereas somatic mutations in other aldosterone-driving genes are rare.

Due to its rare incidence, molecular features of primary aldosteronism (PA) in young adults are largely unknown. Recently developed targeted mutational analysis identified aldosterone-driver somatic mutations in aldosterone-producing lesions, including aldosterone-producing adenomas (APAs), aldosterone-producing nodules (APNs), and aldosterone-producing micronodules, formerly known as aldosterone-producing cell clusters.

Angiotensin II (AngII) stimulates adrenocortical cells to produce aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in the precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. These data support a model in which coordinated increases in transcription and decay facilitate the major transcriptomic changes required to implement a pro-steroidogenic expression program that actively resolved to prevent aldosterone overproduction.

Aldosterone is the mineralocorticoid responsible for sodium retention, thus increased blood volume and pressure. Excessive production of aldosterone results in high blood pressure as well as renal disease, stroke, and visual loss via both direct effects and effects on blood pressure. Weight gain is often associated with increased blood pressure, but it remains unclear how obesity increases blood pressure. Obese patients typically have higher lipoprotein levels; moreover, some studies have suggested that aldosterone levels are also elevated and represent a link between obesity and hypertension. Very-low-density lipoprotein (VLDL) functions to transport triglycerides from the liver to peripheral tissues. Although previous studies have demonstrated that VLDL can stimulate aldosterone production, the mechanisms underlying this effect are largely unclear. Here we show for the first time that phospholipase D (PLD) is involved in VLDL-induced aldosterone production in both a human adrenocortical cell line (HAC15) and primary cultures of bovine zona glomerulosa cells. Our data also reveal that PLD mediates steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2) expression via increasing the phosphorylation (activation) of their regulatory transcription factors. Finally, by using selective PLD inhibitors, our studies suggest that both PLD1 and PLD2 isoforms play an important role in VLDL-induced aldosterone production.

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the TGF-β ligand superfamily. We have identified a functional BMP system equipped with the ligand (BMP4), receptors (BMP type II receptor, BMP type IA receptor, also called ALK3) and the signaling proteins, namely the mothers against decapentaplegic homologs 1, 4, and 5 in the human adrenal gland and the human adrenocortical cell line H295R. Microarray, quantitative RT-PCR, and immunohistochemistry confirmed that BMP4 expression was highest in the adrenal zona glomerulosa followed by the zona fasciculata and zona reticularis. Treatment of H295R cells with BMP4 caused phosphorylation of the mothers against decapentaplegic and a profound decrease in synthesis of the C19 steroids dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione. Administration of BMP4 to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of 17α-hydroxylase/17,20-lyase (CYP17A1 and P450c17, respectively) but no significant effect on the mRNA levels of cholesterol side-chain cleavage cytochrome P450 (CYP11A1) or type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse the negative effects of BMP4 with respect to both CYP17A1 transcription and DHEA secretion in the H295R cell line. Collectively the present data suggest that BMP4 is an autocrine/paracrine negative regulator of C19 steroid synthesis in the human adrenal and works by suppressing P450c17.

The effects of retinoids on adrenal aldosterone synthase gene (CYP11B2) expression and aldosterone secretion are still unknown. We therefore examined the effects of nuclear retinoid X receptor (RXR) pan-agonist PA024 on CYP11B2 expression, aldosterone secretion and blood pressure, to elucidate its potential as a novel anti-hypertensive drug. We demonstrated that PA024 significantly suppressed angiotensin II (Ang II)-induced CYP11B2 mRNA expression, promoter activity and aldosterone secretion in human adrenocortical H295R cells. Human CYP11B2 promoter functional analyses using its deletion and point mutants indicated that the suppression of CYP11B2 promoter activity by PA024 was in the region from -1521 (full length) to -106 including the NBRE-1 and the Ad5 elements, and the Ad5 element may be mainly involved in the PA024-mediated suppression. PA024 also significantly suppressed the Ang II-induced mRNA expression of transcription factors NURR1 and NGFIB that bind to and activate the Ad5 element. NURR1 overexpression demonstrated that the decrease of NURR1 expression may contribute to the PA024-mediated suppression of CYP11B2 transcription. PA024 also suppressed the Ang II-induced mRNA expression of StAR, HSD3β2 and CYP21A2, a steroidogenic enzyme group involved in aldosterone biosynthesis. Additionally, the PA024-mediated CYP11B2 transcription suppression was shown to be exerted via RXRα. Moreover, the combination of PPARγ agonist pioglitazone and PA024 caused synergistic suppressive effects on CYP11B2 mRNA expression. Finally, PA024 treatment significantly lowered both the systolic and diastolic blood pressure in Tsukuba hypertensive mice (hRN8-12 x hAG2-5). Thus, RXR pan-agonist PA024 may be a candidate anti-hypertensive drugs that acts via the suppression of aldosterone synthesis and secretion.

The atrophy and hypofunction of the adrenal cortex following long-term pharmacologic glucocorticoid therapy is a major health problem necessitating chronic glucocorticoid replacement that often prolongs the ultimate return of endogenous adrenocortical function. Underlying this functional recovery is anatomic regeneration, the cellular and molecular mechanisms of which are poorly understood. Investigating the lineage contribution of cortical Sonic hedgehog (Shh)+ progenitor cells and the SHH-responsive capsular Gli1+ cells to the regenerating adrenal cortex, we observed a spatially and temporally bimodal contribution of both cell types to adrenocortical regeneration following cessation of glucocorticoid treatment. First, an early repopulation of the cortex is defined by a marked delamination and expansion of capsular Gli1+ cells, recapitulating the establishment of the capsular-cortical homeostatic niche during embryonic development. This rapid repopulation is promptly cleared from the cortical compartment only to be supplanted by repopulating cortical cells derived from the resident long-term-retained zona glomerulosa Shh+ progenitors. Pharmacologic and genetic dissection of SHH signaling further defines an SHH-dependent activation of WNT signaling that supports regeneration of the cortex following long-term glucocorticoid therapy. We define the signaling and lineage relationships that underlie the regeneration process.

Humans with PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the Prop1(null) (Prop1(-/-)) and the Ames dwarf (Prop1(df/df)) mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism.

Pheochromocytomas (PHEOs) are relatively rare catecholamine-producing tumors derived from adrenal medulla. Tumor microenvironment (TME) including neoangiogenesis has been explored in many human neoplasms but not necessarily in PHEOs. Therefore, in this study, we examined tumor infiltrating lymphocytes (CD4 and CD8), tumor associated macrophages (CD68 and CD163), sustentacular cells (S100p), and angiogenic markers (CD31 and areas of intratumoral hemorrhage) in 39 cases of PHEOs in the quantitative fashion. We then compared the results with pheochromocytoma of the adrenal gland scaled score (PASS), grading system for pheochromocytoma and paraganglioma (GAPP) and the status of intra-tumoral catecholamine-synthesizing enzymes (TH, DDC, and PNMT) as well as their clinicopathological factors. Intratumoral CD8 (p = 0.0256), CD31 (p = 0.0400), and PNMT (p = 0.0498) status was significantly higher in PHEOs with PASS <4 than PASS ≧4. In addition, intratumoral CD8+ lymphocytes were also significantly more abundant in well-than moderately differentiated PHEO according to GAPP score (p = 0.0108) and inversely correlated with tumor size (p = 0.0257). Intratumoral CD68+ cells were significantly higher in PHEOs with regular or normal histological patterns than those not (p = 0.0370) and inversely correlated with tumor size (p = 0.0457). The status of CD163 was significantly positively correlated with that of CD8 positive cells (p = 0.0032). The proportion of intratumoral hemorrhage areas was significantly higher in PHEOs with PASS ≧4 (p = 0.0172). DDC immunoreactivity in tumor cells was significantly positively correlated with PASS score (p = 0.0356) and TH status was significantly higher in PHEOs harboring normal histological patterns (p = 0.0236) and cellular monotony (p = 0.0219) than those not. Results of our present study did demonstrate that abundant CD8+ and CD68+ cells could represent a histologically low-scored tumor. In particular, PHEOs with increased intratumoral hemorrhage should be considered rather malignant. In addition, abnormal catecholamine-producing status of tumor cells such as deficient PNMT and TH and increased DDC could also represent more aggressive PHEOs.

Adequate access to fresh or frozen normal adrenal tissue has been a primary limitation to the enhanced characterization of the adrenal zones via RNA sequencing (RNAseq). Herein, we describe the application of targeted RNAseq to formalin-fixed paraffin-embedded (FFPE) normal adrenal gland specimens. Immunohistochemistry (IHC) was used to visualize and guide the capture of the adrenocortical zones and medulla. Following IHC-based tissue capture and isolation of RNA, high-throughput targeted RNAseq highlighted clear transcriptomic differences and identified differentially expressed genes among the adrenal zones. Our data demonstrate the ability to capture FFPE adrenal zone tissue for targeted transcriptomic analyses. Future comparison of normal adrenal zones will improve our understanding of transcriptomic patterns and help identify potential novel pathways controlling zone-specific steroid production.

Somatic gene mutations that facilitate inappropriate intracellular calcium entrance have been identified in most aldosterone-producing adenomas (APAs). Studies suggest that angiotensin II and adrenocorticotropic hormone (ACTH) augment aldosterone production from APAs. Little is known, however, regarding possible variations in response to hormonal stimuli between APAs with different aldosterone-driver mutations.

Adrenal steroid hormone production is a dynamic process stimulated by adrenocorticotropic hormone (ACTH) and angiotensin II (AngII). These ligands initialize a rapid and robust gene expression response required for steroidogenesis. Here, we compare the predominant human immortalized cell line model, H295R cell, with primary cultures of adult adrenocortical cells derived from human kidney donors. We performed temporally resolved RNA-seq on primary cells stimulated with either ACTH or AngII at multiple time points. The magnitude of the expression dynamics elicited by ACTH was greater than AngII in primary cells. This is likely due to the larger population of adrenocortical cells that are responsive to ACTH. The dynamics of stimulus-induced expression in H295R cells are mostly recapitulated in primary cells. However, there are some expression responses in primary cells absent in H295R cells. These data are a resource for the endocrine community and will help researchers determine whether H295R is an appropriate model for the specific aspect of steroidogenesis that they are studying.

Aldosterone production is physiologically under the control of circulating potassium and angiotensin II as well as adrenocorticotropic hormone and other secretagogues such as serotonin. The adrenal's capacity to produce aldosterone relies heavily on the expression of a single enzyme, aldosterone synthase (CYP11B2). This enzyme carries out the final reactions in the synthesis of aldosterone and is expressed almost solely in the adrenal zona glomerulosa. From a disease standpoint, primary aldosteronism (PA) is the most common of all adrenal disorders. PA results from renin-independent adrenal expression of CYP11B2 and production of aldosterone. The major causes of PA are adrenal aldosterone-producing adenomas (APA) and adrenal idiopathic hyperaldosteronism. Our understanding of the genetic causes of APA has significantly improved through comprehensive genetic profiling with next-generation sequencing. Whole-exome sequencing has led to the discovery of mutations in six genes that cause renin-independent aldosterone production and thus PA. To facilitate broad-based prospective and retrospective studies of APA, recent technologic advancements have allowed the determination of tumor mutation status using formalin-fixed paraffin-embedded (FFPE) tissue sections. This approach has the advantages of providing ready access to archival samples and allowing CYP11B2 immunohistochemistry-guided capture of the exact tissue responsible for inappropriate aldosterone synthesis. Herein we review the methods and approaches that facilitate the use of adrenal FFPE material for DNA capture, sequencing, and mutation determination.

Graves' disease (GD) is an autoimmune disorder caused by autoantibodies against the thyroid stimulating hormone receptor (TSHR) leading to overstimulation of the thyroid gland. Thyroid eye disease (TED) is the most common extra thyroidal manifestation of GD. Therapeutic options to treat TED are very limited and novel treatments need to be developed. In the present study we investigated the effect of linsitinib, a dual small-molecule kinase inhibitor of the insulin-like growth factor 1 receptor (IGF-1R) and the Insulin receptor (IR) on the disease outcome of GD and TED.