Project description:H295R human adrenocortical cells treated with or without angiotensin II (100 nM) for 3 hr. Keywords = adrenal angiotensin aldosterone Keywords: parallel sample
Project description:H295R human adrenocortical cells treated with or without angiotensin II (100 nM) for 3 hr. Keywords = adrenal angiotensin aldosterone
Project description:Angiotensin II (Ang-II) regulates adrenal steroid production and gene transcription through several signaling pathways. Changes in gene transcription occur within minutes after Ang-II stimulation, causing an acute increase in aldosterone production and subsequent increase in the overall capacity to produce aldosterone. Our goal was to compare the Ang-II regulation of early gene expression and confirm the upregulation of selected genes using quantitative real-time RT-PCR (qPCR) across three species: human, bovine, and rat. Experiment Overall Design: Microarray analysis was performed using samples from control and Ang-II-(10 nM) treated (1 hour) cells from human adrenocortical tumor cell line H295-R, and primary adrenal glomerulosa cells from bovine and rat, applied respectively to human HG-133 + 2 , bovine, and rat 230-2 Affymetrix chips. qPCR was performed to confirm upregulation of selected genes using mRNA. Dye Swap was not used. Human samples (H295R) include 3 replicates of Basal (controls) and 3 repeats of Angiotensin II samples. Human H295R cells include also a group of cycloheximide (protein synthesis blocker) and angiontensin II + cycloheximide in order to check if the genes were direct targets of angiotensin II.
Project description:H295R human adrenocortical cells were cultured in H295R complete media containing DMEM:F12 (1:1) supplemented with 2% Ultroser G (Biosepra, Villeneuve-la-Garenne, France), ITS-Plus (Discovery Labware, Bedford, MA) and an antibiotic/antimycotic mixture (Invitrogen, Carlsbad, CA) in 175 cm2 flasks until subconfluente. Media was replaced with 40 ml fresh media containing different agents and cultured for 3 h. Treatments: Angiotensin II (100 nM), potassium (16 mM) and forskolin (10 uM). Total RNA extracted with RNAesy Midi Kit (Qiagen) with on-column DNase digestion Experiment Overall Design: Controls, n=2 Experiment Overall Design: Angiotensin=1 Experiment Overall Design: Potassium=1 Experiment Overall Design: Forskolin=1 Experiment Overall Design: Same biological sample used for GPL96 and GPL97
Project description:H295R human adrenocortical cells were cultured in H295R complete media containing DMEM:F12 (1:1) supplemented with 2% Ultroser G (Biosepra, Villeneuve-la-Garenne, France), ITS-Plus (Discovery Labware, Bedford, MA) and an antibiotic/antimycotic mixture (Invitrogen, Carlsbad, CA) in 175 cm2 flasks until subconfluente. Media was replaced with 40 ml fresh media containing different agents and cultured for 3 h. Treatments: Angiotensin II (100 nM), potassium (16 mM) and forskolin (10 uM). Total RNA extracted with RNAesy Midi Kit (Qiagen) with on-column DNase digestion Keywords: Agent response
Project description:Transcription factor 21 (TCF21) directly binds and regulates SF1 in tumor and normal adrenocortical cells, and both are involved in the development and steroidogenesis of the adrenal cortex. TCF21 is a tumor suppressor gene and its expression is reduced in malignant tumors. In adrenocortical tumors, it is less expressed in adrenocortical carcinomas (ACC) than in adrenocortical adenomas (ACA) and normal tissue. However, a comprehensive analysis to identify TCF21 targets have not yet been conducted in any type of cancer. In this study, we performed Chromatin Immunoprecipitation and Sequencing (ChIP-Seq) in adrenocortical carcinoma cell line (NCI-H295R) overexpressing TCF21, with the aim of identifying TCF21 new targets. The five most frequently identified sequences corresponded to the PRDM7, CNTNAP2, CACNA1B, PTPRN2 and KCNE1B genes. Validation experiments showed that, in NCI-H295R cells, TCF21 regulates gene expression positively in PRDM7 and negatively in CACNA1B. Recently, it was observed that the N-type calcium channel v2.2 (Cav2.2) encoded by CACNA1B gene is important in Angiotensin II signal transduction for corticosteroid biosynthesis in NCI-H295R adrenocortical carcinoma cells. Indeed, TCF21 inhibits CACNA1B and Cav2.2 expression in NCI-H295R. In addition, in a cohort of 55 adult patients with adrenocortical tumor, CACNA1B expression was higher in ACC than ACA, and was related to poor disease-free survival in ACC patients. These results suggest a mechanism of steroidogenesis control by TCF21 in adrenocortical tumor cells, in addition to the control observed through SF1 inhibition. Importantly, steroid production could impair tumor immunogenicity, contributing to the immune resistance described in adrenal cancer.
Project description:RNA sequencing provides a transcriptome-wide view of what processes are activated and repressed during steroidogenesis. Here we performed an RNA-seq time series on primary human adrenocortical cells and H295R cells stimulated with ACTH or AngII (forskolin for H295R). We found that the ligand-induced changes in gene expression largely involved the same genes and similar timing. While stimulus-induced expression changes in H295R cells are recapitulated by primary cells, there are also expression changes unique to primary cells.
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.