Transcriptomic Response Dynamics of Human Primary and Immortalized Adrenocortical Cells to Steroidogenic Stimuli

Figures

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  Figure 1. Steroidogenic expression responses of primary human adrenocortical cells to adrenocorticotropic hormone (ACTH) or angiotensin II (AngII). (A) Primary cell culture schema: (1) Adrenal gland was coincidentally removed from a deceased human kidney donor. (2) Cortical cells were isolated, dissociated and then (3) plated in triplicate and allowed to reach confluence before being (4) stimulated with ACTH (purple), AngII (green), or left untreated for basal (gray). (5) RNA was purified from cells stimulated for 3, 6, 12, 24, and 48 h. (6) RNA sequencing libraries were prepared after poly(A) selection. (B) Comparison of normalized responses for ACTH (left) and AngII (right). Heatmap of log2 fold changes in RNA expression between stimulated and unstimulated cells. Red denotes increased expression while blue shows decreased expression. Genes are categorized by pathway.

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  Figure 2. Comparison of transcriptome-wide expression response between ACTH and Ang II in primary adrenocortical cells (A) Principal components (PC) analysis of all stimulation treatments and time points mean expression of replicates for all expressed genes. Points are colored by stimulation treatment; ACTH (purple), AngII (green), or left untreated for basal (gray). (B) Euler diagram of the genes that were differentially expressed at any time point in response to ACTH (purple) compared to AngII (green) and their intersection (gray). (C) Scatterplot of expression changes induced by ACTH (X axis) or AngII (Y axis) for matching time points. (D) Barplot showing the correlation of gene expression changes induced by AngII and ACTH by each time point. The red bars denote the matched time point for either stimulation treatment.

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  Figure 3. Comparison of ACTH- and AngII-induced gene expression hallmarks in primary cells. (A) For each treatment condition, normalized enrichment scores (NES) were calculated by Gene Set Enrichment Analysis against Molecular Signatures Database (MSigDB) hallmark gene sets, ordering genes by fold change compared to basal culture. Yellow denotes increased enrichment while blue shows depletion. Hallmark gene sets with no significant (q ≤ 0.05) enrichment or depletion in any condition were excluded from the plot. (B) Line plot of log2 fold change in expression of NPC1 upon treatment with ACTH (left) and AngII (right). Error bars are standard deviation.

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  Figure 4. Projection of AngII-induced H295R differential expression gene clusters onto ACTH- and AngII-induced primary cell expression changes. For each primary cell treatment time point (ACTH left, AngII right), normalized enrichment scores (NES) were calculated by Gene Set Enrichment Analysis against AngII-H295R gene sets determined by temporal and amplitude clustering as reported previously, ordering genes by fold change compared to basal culture. Yellow denotes enrichment while blue shows depletion. H295R AngII gene sets were named by their profile for: direction-activation (a) or repression (r), magnitude-strong (s) or weak (w), and timing-numbered from early to late peak response (1 earliest to peak).

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  Figure 5. Comparison of stimulus-induced expression changes between primary adrenocortical cells and H295R cells. (A) Euler diagram of differentially expressed genes in primary cells stimulated with ACTH (dark purple) as compared to differentially expressed genes in H295R cells stimulated with forskolin (light purple) and their intersection (gray). (B) Euler diagram of differentially expressed genes in primary cells stimulated with AngII (dark green) as compared to differentially expressed genes in H295R cells stimulated with AngII (light green) and their intersection (gray). (C) Line plot of log2 fold change in expression upon treatment with ACTH or forskolin (left) and AngII (right). For primary cells, log2 fold changes were calculated versus basal media at matching times, whereas for H295R cells, log2 fold changes were calculated versus unstimulated cells. Black lines represent log2 fold changes of primary cells and red lines represent log2 fold changes of H295R cells. Error bars are standard error. An asterisk by primary or H295R indicates whether the gene exhibited statistically significant differential expression (see methods for details).