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Lats1/2 depletion in male murine steroidogenic zG adrenocortical cells

ABSTRACT: In order to understand the role of Hippo pathway in mice adrenal glands, we inactivated lats1/2 in a transgenic mouse model using the cre recombinase system in aldosterone-producing zG cells. RNAseq analysis on whole adrenal glands were performed on male mice

ORGANISM(S): Mus musculus

PROVIDER: GSE227351 | GEO | 2024/12/30

REPOSITORIES: GEO

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Project description:In order to analyze the transcriptome characteristics of aldosterone producing cell clusters (APCC) we compared transcript abundances of APCC, zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors. The frozen adrenal glands in O.C.T. compound were cut into 7um sections, and every 10-th section immunostained for aldosterone synthase (CYP11B2). The remaining sections were stained with cresyl violet and used for laser-capture microdissection of tissue to use in the array assays. APCC and ZG samples were captured from CYP11B2 positive regions based on the CYP11B2-stained sections. ZF and ZR were captured from lipid-rich cells in the middle layer and compact cells outside of the medulla, respectively. RNA was isolated using PicoPure RNA isolation kits (Molecular Devices, Sunnyvale, CA). 1-10 ng total RNA was reverse-transcribed and amplified with the Ovation Pico WTA System V2 (NuGEN Technologies, San Carlos, CA). cDNA was purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and biotin-labeled using Encore Biotin Module (NuGEN Technologies), followed by hybridization to GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). Expression values were calculated using the robust multi-array average method (RMA). This resulted in base-2 log-transformed data for each of the 4 tissues from each of the 4 people. In addition to the raw and processed data we also supply a supplementary Excel file holding the data and some statistical analysis, which has features to make simple graphs, and holds probe-set annotation that we used at that time (users may wish to obtain new annotation though). We fit two-way ANOVA models with terms for 4 tissues and 4 people, and compared each probe-set between every pair of tissues using F-tests for pairwise contrasts. We modeled people effects since they were not negligible. The supplement shows how to calculate the tests. Aldosterone producing cell clusters (APCC), zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors, were individually assayed on 16 Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Expression values were estimated with the Robust Multi-Arry average (RMA) algorithm, which resulted in log-2 transformed values for each of 54675 probe sets.

Project description:Steroidogenic acute regulatory protein (Star) facilitates cholesterol transfer into the inner mitochondrial membrane in the acute and regulated production of steroid hormones. Mice lacking Star (Star-/-) share the phenotypes with patients with congenital lipoid adrenal hyperplasia such as compromised production of steroid hormones and florid accumulation of cholesterol esters in adrenal glands and gonads. To define specific patterns of molecular changes with disruption of Star, we performed a transcriptome analysis of steroidogenic cells in adrenal glands. We harvested adrenal glands at E17.5 or 18.5 from seven wild-type (Star+/+) mice or four Star-/- mice having the transgene targeting enhanced green fluorescent protein (eGFP) under the control of regulatory sequences of mouse Star gene. Steroidogenic cells were selectively isolated by fluorescent-activated cell sorting. The gene expression profile of the fluorescence-positive cells was obtained with Agilent Whole Mouse Genome Microarray and was confirmed by quantitative real-time PCR. We identified 961 and 622 genes that were significantly up-regulated and down-regulated, respectively, in Star-/- mice compared with Star+/+ mice (fold difference ≥2, p value of Student t test <0.05, and Benjamini-Hochberg false discovery rate of multiple comparison test <0.2). In Star-/- mice, expression levels of genes involved in cholesterol mobilization and efflux or immune response as antigen presenting cells were significantly increased, and transition from fetal to adult adrenocortical cells were significantly decreased, whereas those of genes related to steroid hormone biosynthesis or cholesterol biosynthesis and influx were not significantly changed. The trancriptome analysis revealed hitherto undescribed characteristic changes in adrenocortical cells and expanded our understanding of the pathophysiology of the steroidogenic cells with disruption of Star. Gene expression profiles of isolated adrenal steroidogenic cells were compared between seven wild-type mice and four knockout mice lacking steroidogenic acute regulatory protein at E17.5-18.5.