TPD52 Inhibits Aldosterone Synthesis Through Suppression of CAMKK2 Signaling
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE319287 | GEO | 2026/02/22
REPOSITORIES: GEO
Dataset's files
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| GSE319287_FPKMs_All_Samples.txt.gz | Txt |
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Project description:Background. Primary aldosteronism is the most common form of secondary hypertension. The most frequent genetic cause of aldosterone producing adenoma (APA) are somatic mutations in the potassium channel KCNJ5. They affect the ion selectivity of the channel, with sodium influx leading to cell membrane depolarization and activation of calcium signalling, the major trigger for aldosterone biosynthesis. Methods. To investigate how KCNJ5 mutations lead to the development of APA, we established an adrenocortical cell model in which sodium entry into the cells can be modulated “on demand” using chemogenetic tools (H295R-S2 α7-5HT3 cells). We investigated their functional and molecular characteristics with regard to aldosterone biosynthesis and cell proliferation. Results. A clonal cell line with stable expression of the chimeric α7-5HT3 receptor in H295R-S2 cells was obtained. Increased sodium entry through the α7-5HT3 receptor upon stimulation with uPSEM-817 led to cell membrane depolarization, opening of voltage-gated Ca2+ channels and increased intracellular Ca2+ concentrations, resulting in the stimulation of CYP11B2 expression and increased aldosterone biosynthesis. Increased intracellular sodium influx did not increase proliferation, but rather induced apoptosis. RNA sequencing and steroidome analyses revealed unique profiles associated with Na+ entry, with only partial overlap with angiotensin II or potassium induced changes. Conclusion. H295R-S2 α7-5HT3 cells are a new model reproducing the major features of cells harbouring KCNJ5 mutations. Increased expression of CYP11B2 and stimulation of the mineralocorticoid biosynthesis pathway are associated with a decrease of cell proliferation and an increase of apoptosis, indicating that additional events may be required for the development of APA.
2025-01-19 | GSE282358 | GEO
Project description:To investigate the role of NFATc4 in aldosterone production, we created NFATc4 knock-out NCI-H295R cell line and overexpressed active NFATc4 in wild type NCI-H295R cells. We then analyzed the transcriptome of the four cell types.
2023-05-12 | GSE225735 | GEO
Project description:Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in KCNJ5 (APA-KCNJ5MUT) are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type KCNJ5 (APA-KCNJ5WT). Here, we used spatial transcriptomics profiling of adrenal tissue cryosections to define the role of transcriptomic reprogramming in APA pathophysiology. Our findings advance the understanding of the transcriptional context of inter- and intra-tumoral APA heterogeneity and provide novel insight into the genotype-dependent tumor expansion capabilities of APAs.
2024-08-13 | GSE274314 | GEO
Project description:The mineralocorticoid hormone, aldosterone, is secreted by the adrenal zona glomerulosa (ZG) in response to high plasma K+ and hypovolemia and promotes renal Na+ reabsorption and K+ secretion. Hence, the regulation of aldosterone secretion is critical for the control of ion homeostasis and blood pressure. While the kinase pathways regulating aldosterone production are well studied, little is known about the involved phosphatases. Using the human adrenocortical carcinoma cell line NCI-H295R, we found that the mRNA expression of the aldosterone synthase increases significantly within 6 hours after K+ exposure. This increase was inhibited in a dose-dependent manner by the calcineurin inhibitors tacrolimus and cyclosporine A. Calcineurin (Cn) is a serine-threonine-specific, Ca2+ and CaM-activated protein phosphatase essential for lymphocyte, neuronal and cardiac function. The physiologic role of Cn in the ZG cells and the molecular pathways by which Cn regulates the K+-stimulated secretion of aldosterone are unknown. To answer these questions, we stimulated NCI-H295R cells with K+ with or without Tacrolimus and studied the phosphorylation pattern of cytoplasmic proteins by phospho-proteomics. We generated a map of the changes in the Ser/Thr phosphorylation in adrenocortical cells upon stimulation with K+ and identified Cn-regulated phosphoproteins.
2023-05-25 | PXD027856 | Pride
Project description:Analysis of aldosterone-producing adenoma (APA) samples from patients with primary hyperaldosteronism. These APAs have a somatic mutation in either KCNJ5, CACNA1D, or ATP1A1. Results provide insight into the different mechanisms each mutation may cause leading to elevated aldosterone production in APA. In this dataset, we include expression data from aldosterone-producing adenomas (APAs) with a somatic mutation in either KCNJ5, CACNA1D, or ATP1A1. These data are used to obtain differentially expressed genes between KCNJ5 mutant APAs and CACNA1D/ATP1A1 mutant APAs. A total of 13 samples were analyzed (8 KCNJ5 mutant APAs and 5 CACNA1D/ATP1A1 mutant APAs). 43 genes had a false discovery rate (FDR) <0.5% and were >2-fold different between KCNJ5 mutant APAs and 5 CACNA1D/ATP1A1 mutant APAs. The two sets of genotypes were separated on unsupervised hierarchical clustering of 1475 genes correlating >0.6 with CYP11B2.
2013-07-14 | E-GEOD-48303 | biostudies-arrayexpress
Project description:Primary aldosteronism (PA), the most common form of endocrine hypertension, is characterized by inappropriately elevated aldosterone production via renin-independent mechanisms. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2)-guided sequencing approach. Herein, using CYP11B2-guided whole-exome sequencing (WES) and targeted amplicon sequencing, we detected two closely-stationed somatic variants in SLC30A1 in five APAs (p.L51_A57del, n=3; p.L49_L55del, n=2) that were devoid of any of the known aldosterone-driver mutations. SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). PA cases with SLC30A1 mutations showed male dominance and demonstrated increased serum aldosterone concentration compared with age-matched male controls. We tested functional effects of the variant SLC30A1L51_A57del in a doxycycline-inducible system using the human adrenocortical HAC15 cell line. Functional in vitro studies following doxycycline treatment indicated increased adrenal cell aldosterone production, CYP11B2 mRNA expression, CYP11B2 promoter activity, depolarization of the resting membrane potential and increased cytosolic Ca2+ levels in the SLC30A1L51_A57del cells. Collectively, these data support a pathological role for mutant SLC30A1 on the development of PA.
2023-07-03 | GSE236437 | GEO
Project description:Primary aldosteronism (PA) is the most common endocrine hypertension comprising 10% of hypertensive patients. A recent series of transcriptome analyses using DNA microarray has shown that neumerous genes are differentially expressed between aldosterone-producing adenoma (APA) and its adjacent adrenal gland (AAG) tissue from the same patient. However, the molecular mechanism(s) of pathogenesis of APA has not yet been fully clarified. Although growing body of evidence has shown that epigenetic abnormalities including DNA methylation play a key role in tumorigenesis, there are few studies with regard to DNA methylation in APA. In the present study, to elucidate the pathogenic relationship between gene expression and DNA methylation in PA, we conducted an integrated analysis of transcriptome and methylome data for paired APA-AAG samples from the same patient. Adrenal specimens were obtained from 7 Japanese patients with APA, who underwent adrenalectomy at Tokyo Medical and Dental University. RNA and DNA samples were extracted from those 7 paired APA and AAG tissues. Gene expression and genome-wide DNA methylation profiles were obtained using SurePrint G3 Human GE 8x60K Microarray (Agilent) and Infinium HumanMethylation450 Beadchip (Illumina), respectively. Transcriptome anlaysis identified 244 significantly (2 fold<) upregulated genes in APA compared to AAG. The upregulated genes include the previously studied genes such as PCP4, ALDH1A2, and CYP11B2, and other genes that have not been previously studied, such as HOXA9, HOXA11, and HOXB9. Gene ontology (GO) analysis for these upregulated genes identified the calcium signaling pathway to be most significantly enriched with the upregulated genes (8 genes). Methylome analysis revealed that APA was globally hypomethylated compared to AAG regardless of gene feature groups, namely, TSS1500, TSS200, 5’UTR, 1stExon, gene body, and 3’UTR. GO analysis for the genes showing hypomethylation at TSS1500/TSS200 regions in APA identified the term “cytokine-cytokine receptor interaction” to be most significantly enriched with hypomethylated genes (37 genes). Integrated analysis of gene expression and genome-wide DNA methylation profiles identified 18 genes that are upregulated and whose TSS1500/TSS200 regions are hypomethylated in APA compared to AAG. These genes include CYP11B2 and MC2R (ACTH receptor). In conclusion, this is the first genome-wide study for PA that integrated transcriptome and methylome data. Global DNA hypomethylation in APA and concordant transcriptional up-regulation of some key genes, such as CYP11B2 and MC2R, may play crucial roles in the pathophysiological significance in PA. Adrenal specimens were obtained from 7 Japanese patients with APA, who underwent adrenalectomy at Tokyo Medical and Dental University. RNA and DNA samples were extracted from those 7 paired APA and AAG tissues. Gene expression and genome-wide DNA methylation profiles were obtained using SurePrint G3 Human GE 8x60K Microarray (Agilent) and Infinium HumanMethylation450 Beadchip (Illumina), respectively.
2015-05-09 | E-GEOD-60043 | biostudies-arrayexpress
Project description:Primary aldosteronism (PA) is the most common endocrine hypertension comprising 10% of hypertensive patients. A recent series of transcriptome analyses using DNA microarray has shown that neumerous genes are differentially expressed between aldosterone-producing adenoma (APA) and its adjacent adrenal gland (AAG) tissue from the same patient. However, the molecular mechanism(s) of pathogenesis of APA has not yet been fully clarified. Although growing body of evidence has shown that epigenetic abnormalities including DNA methylation play a key role in tumorigenesis, there are few studies with regard to DNA methylation in APA. In the present study, to elucidate the pathogenic relationship between gene expression and DNA methylation in PA, we conducted an integrated analysis of transcriptome and methylome data for paired APA-AAG samples from the same patient. Adrenal specimens were obtained from 7 Japanese patients with APA, who underwent adrenalectomy at Tokyo Medical and Dental University. RNA and DNA samples were extracted from those 7 paired APA and AAG tissues. Gene expression and genome-wide DNA methylation profiles were obtained using SurePrint G3 Human GE 8x60K Microarray (Agilent) and Infinium HumanMethylation450 Beadchip (Illumina), respectively. Transcriptome anlaysis identified 244 significantly (2 fold<) upregulated genes in APA compared to AAG. The upregulated genes include the previously studied genes such as PCP4, ALDH1A2, and CYP11B2, and other genes that have not been previously studied, such as HOXA9, HOXA11, and HOXB9. Gene ontology (GO) analysis for these upregulated genes identified the calcium signaling pathway to be most significantly enriched with the upregulated genes (8 genes). Methylome analysis revealed that APA was globally hypomethylated compared to AAG regardless of gene feature groups, namely, TSS1500, TSS200, 5’UTR, 1stExon, gene body, and 3’UTR. GO analysis for the genes showing hypomethylation at TSS1500/TSS200 regions in APA identified the term “cytokine-cytokine receptor interaction” to be most significantly enriched with hypomethylated genes (37 genes). Integrated analysis of gene expression and genome-wide DNA methylation profiles identified 18 genes that are upregulated and whose TSS1500/TSS200 regions are hypomethylated in APA compared to AAG. These genes include CYP11B2 and MC2R (ACTH receptor). In conclusion, this is the first genome-wide study for PA that integrated transcriptome and methylome data. Global DNA hypomethylation in APA and concordant transcriptional up-regulation of some key genes, such as CYP11B2 and MC2R, may play crucial roles in the pathophysiological significance in PA. Adrenal specimens were obtained from 7 Japanese patients with APA, who underwent adrenalectomy at Tokyo Medical and Dental University. RNA and DNA samples were extracted from those 7 paired APA and AAG tissues. Gene expression and genome-wide DNA methylation profiles were obtained using SurePrint G3 Human GE 8x60K Microarray (Agilent) and Infinium HumanMethylation450 Beadchip (Illumina), respectively.
2015-05-09 | E-GEOD-60042 | biostudies-arrayexpress
Project description:Recent advances in omics techniques have allowed detailed genetic characterization of aldosterone-producing adenoma (APA). The pathogenesis of APA is characterized by tumorigenesis-associated aldosterone synthesis. The pathophysiological intricacies of APAs have not yet been elucidated at the level of individual cells . Therefore, a single-cell level analysis is speculated to be valuable in studying the differentiation process of APA. We conducted single-nuclei RNA sequencing (snRNA-seq) of APAs and non-functional adenomas (NFA) obtained from three and two patients, respectively.The snRNA-seq revealed the intratumoral heterogeneity of APA and identified cell populations consisting of a shared cluster of NFA and APA. In addition, we extracted two cell differentiation pathways in APA and obtained a cell population specialized in aldosterone synthesis. Genes related to ribosomes and neurodegenerative diseases were upregulated in one of these pathways, whereas those related to the regulation of actin cytoskeleton were upregulated in the other pathway. Furthermore, the total RNA reads in the nucleus were higher in highly differentiated clusters, indicating a marked activation of transcription per cell. The snRNA-seq provides novel differentiation pathways within APA tumors, which will further support our understanding of its pathophysiology including endocrine function and tumorigenesis.
2023-12-01 | GSE242404 | GEO
Project description:The source of aldosterone in 30 to 40 % of patients with primary hyperaldosteronism (PA) is unilateral aldosterone-producing adenoma (APA). The mechanisms causing elevated aldosterone production in APA are unknown. Herein, we examined expression of G-protein coupled receptors (GPCR) in APA and demonstrate that compared to normal adrenals there is a general elevation of certain GPCR in many APA and/or ectopic expression of GPCR in others. RNA samples from normal adrenals (n = 5), APAs (n = 10), and cortisol-producing adenomas (CPAs) (n=13) were used on 15 genomic expression arrays, each of which included 223 GPCR transcripts presented in at least one out of 15 of the independent microarrays. The array results were confirmed using real-time RT-PCR (qPCR). Four GPCR transcripts exhibited a statistically significant increase that was greater than 3-fold compared to normal adrenals, suggesting a general increase in expression compared to normal adrenal glands. Four GPCR transcripts exhibited a greater than 15-fold increase of expression in one or more of the APA samples compared to normal adrenals. qPCR analysis confirmed array data and found the receptors with the highest fold increase in APA expression to be luteinizing hormone receptor (LH-R), serotonin receptor 4 (HTR4), gonadotropin-releasing hormone receptor (GnRHR), glutamate receptor metabotropic 3 (GRM3), endothelin receptor type B-like protein (GPR37), and ACTH receptor (MC2R). There are also sporadic increased expressions of these genes in the CPAs. Together, these findings suggest a potential role of altered GPCR expression in many cases of PA and provide candidate GPCR for further study. Keywords: disease state analysis Normal human adult adrenals were obtained through the Cooperative Human Tissue Network (Philadelphia, PA) and Clontech (Palo Alto, CA). All the normal adrenal samples came from patients that underwent adrenalectomy along with a renalectomy due to renal carcinoma. The adrenal samples acquired from autopsies were obtained no more than six hours after death, and it was confirmed that the causes of death were unrelated to adrenal function. Real-time quantitative RT-PCR was used to check the legitimacy of the normal control adrenal samples. Examination of tissue sections from each adrenal gland also suggested normal histology. The APA samples were obtained from the Departments of Medicine at UT Southwestern and Division of Endocrinology at the University of Padua. All APA samples were from Conn’s Syndrome patients with significantly elevated circulating aldosterone that was lowered to the normal range after surgical removal of the tumor. Thirty-two adenomas were collected from patients with primary hyperaldosteronism. Twenty-eight of these adenomas had levels of CYP11B2 that were two standard deviations (SD) greater than seen in normal adrenal glands and these samples were used for analyses. It is currently not clear if the adenomas with low CYP11B2 expression are the source of aldosterone in these patients. Other studies suggest that subcapsular micronodules have varying expression of steroidogenic enzymes-some with aldosterone synthase and some without (Shigematsu et al., 2006). Future studies are required to determine if some tumor-bearing adrenals from primary hyperaldosteronsim patients also have small CYP11B2 positive micronodules as the source of aldosterone production. The cortisol-producing adenoma (CPA) tissues were obtained from Division of Endocrinology at the University of Padua from patients with Cushing’s syndrome. Each of the patients was cured following surgical removal of the adenoma. The use of these tissues was approved by the Institutional Review Boards of the University of Texas Southwestern Medical Center (Dallas, TX), the Medical College of Georgia (Augusta, GA), and University of Padua (Italy). In addition, this study was approved by the ethical committee at the University of Padua, and informed consent was obtained from every patient. Microarray Analysis Total RNA isolated from normal adult adrenal glands (n = 5) and APA (n = 10) were used on 15 genomic expression arrays. In brief, RNA was hybridized to an Affymetrix human HG-U133+2 oligonucleotide microarray set containing 54,675 probe sets representing approximately 40,500 independent human genes. The arrays were scanned at high resolution using an Affymetrix GeneChip Scanner 3000 located at Medical College of Georgia Microarray Core Facility (Augusta, GA). Results were analyzed using GeneSpring GX 7.3.1 software (Silicon Genetics, Redwood City, CA) to identify differences in expression of GPCR between normal adult adrenal and APA. RNA Extraction The tissue was pulverized in liquid nitrogen to a powder. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA). The purity and integrity of the RNA were assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and its quantity was determined by NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA Synthesis 2 µg of total RNA was reverse transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) following manufacturer recommendations and incubated at 25 ºC for 10 min, and 37 ºC for 2 h. The synthesized cDNA was subjected to 1:10 dilution and stored at -20ºC. Real-Time Quantitative PCR Primers and probes for the amplification of the selected GPCR sequences were performed using the TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) based on published sequences for genes encoding the respective human GPCR. The gene symbols and AB assay numbers are listed in Table 1. The primer and probe set for human CYP11B2 was designed using Primer Express 3.0 (Applied Biosystems, Foster City, CA) and purchased from IDT (Integrated DNA Technologies, Inc, IA) as published previously (Saner-Amigh et al., 2006). In brief: Forward: 5’-GGCAGAGGCAGAGATGCTG-3’, Reverse: 5’- CTTGAGTTAGTGTCTCCACCAGGA-3’, Probe: 5’-CTGCACCACGTGCTGAAGCACT-3’. PCR reactions were performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) with a total volume of 20 μl per reaction following the reaction parameters recommended by the manufacturer, which includes denaturation at 95ºC for 20 s followed by amplification for 40 cycles (95ºC at 3 s, 60ºC at 30 s, fluorescence measurement). For each GPCR the 20 μl total volume consisted of 10 µl TaqMan Fast Universal PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 900 nM of each primer, 400 nM of the probe and 5 μl of each first-strand cDNA sample. CYP11B2 reaction mix consisted of 10µl TaqMan PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 100 nM of primer/probe mix and 5 μl of each first-strand cDNA sample. 18s rRNA was detected and quantified using the TaqMan Ribosomal RNA Control Reagents (Vic Probe) (Applied Biosystems, Foster City, CA). Each reaction included 10 µl of TaqMan PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 100 nM probe and 50 nM primers. Negative controls contained water instead of first-strand cDNA. Quantitative normalization of cDNA in each tissue-derived sample was performed using expression of 18s rRNA as an internal control. The generated Ct value of each gene was normalized by its respected Ct value of 18s rRNA (∆Ct). Then each gene was further normalized using the average ∆Ct value of the normal adult adrenal (∆∆Ct). The final fold-expression changes were calculated using the equation 2-∆∆Ct (Livak & Schmittgen, 2001). Statistical Analysis Data were analyzed and compared to control values (mean of normal adrenal samples) using the Mann-Whitney Rank Sum test with the SigmaStat 3.0 software package (SPSS, Chicago, IL). Results were considered significantly different when p value was 0.05.
2007-07-18 | E-GEOD-8514 | biostudies-arrayexpress