PPARG dysregulation as a potential molecular target in adrenal Cushing's syndrome

Abstract

Background: We performed a transcriptomic analysis of adrenal signaling pathways in various forms of endogenous Cushing's syndrome (CS) to define areas of dysregulated and druggable targets.

Methodology: Next-generation sequencing was performed on adrenal samples of patients with primary bilateral macronodular adrenal hyperplasia (PBMAH, n=10) and control adrenal samples (n=8). The validation groups included cortisol-producing adenoma (CPA, n=9) and samples from patients undergoing bilateral adrenalectomy for Cushing's disease (BADX-CD, n=8). In vivo findings were further characterized using three adrenocortical cell-lines (NCI-H295R, CU-ACC2, MUC1).

Results: Pathway mapping based on significant expression patterns identified PPARG (peroxisome proliferator-activated receptor gamma) pathway as the top hit. Quantitative PCR (QPCR) confirmed that PPARG (l2fc<-1.5) and related genes - FABP4 (l2fc<-5.5), PLIN1 (l2fc<-4.1) and ADIPOQ (l2fc<-3.3) - were significantly downregulated (p<0.005) in PBMAH. Significant downregulation of PPARG was also found in BADX-CD (l2fc<-1.9, p<0.0001) and CPA (l2fc<-1.4, p<0.0001). In vitro studies demonstrated that the PPARG activator rosiglitazone resulted in decreased cell viability in MUC1 and NCI-H295R (p<0.0001). There was also a significant reduction in the production of aldosterone, cortisol, and cortisone in NCI-H295R and in Dihydrotestosterone (DHT) in MUC1 (p<0.05), respectively.

Outcome: This therapeutic effect was independent of the actions of ACTH, postulating a promising application of PPARG activation in endogenous hypercortisolism.

Keywords: adrenocortical cell line; cortisol; hypercortisolism; primary bilateral macronodular hype; rosiglitazone; steroidome; transcriptome.

Figure 1

Differentially expressed significant genes in NGS between in adrenals of PBMAH vs. controls. Volcano plot showing the relationship between fold change (log2foldchange) and statistical significance (-log10pvalue). The red points represent significantly upregulated genes while blue points represent significantly downregulated genes. The top 32 altered genes are labelled (24 downregulated and 10 upregulated).

Figure 2

Pathway analyses of the significant genes from NGS. The significantly expressed genes were shortlisted (log2FC > abs (2), p<0.05). (A) The shortlisted genes were used for KEGG pathway mapping using ShinyGO online analyses tool. (A) Barplot representation of the top significant pathway hits. Higher fold enrichment refers to higher representation of the genes in the pathway. The pathways are sorted by FDR, with higher significant pathways at the top. (B) Hierarchical clustering of significant pathways: The pathway genes were clustered based on the genes shared amongst the different pathways.

Figure 3

QPCR analyses of the significant genes from pathway analyses. Expression analysis of the pathway genes from PPARG signaling. Data are represented as mean ± standard error of mean (SEM) of -dCT values. Housekeeping gene: Ppia. *p-value <0.05 and FDR<0.05. PBMAH, Primary Bilateral Macronodular Hyperplasia.

Figure 4

QPCR analyses of the significantly altered PPARG pathway genes in CS subtypes. The expression of significantly altered PPARG (A) and its target genes ADIPOQ (B), FABP4 (C), PLIN1 (D) were tested in CS subtypes of CD and CPA including additional controls of APA and normal adrenals. Data are represented as mean ± SEM of -dCT values. Housekeeping gene: Ppia. *p-value <0.05 and FDR<0.05. APA, Aldosterone producing adenoma; CPA, cortisol producing adenoma; BADX-CD, Bilateral adrenalectomized patients with persistent Cushing’s Disease.

Figure 5

Pparg expression in ACTH stimulated murine adrenals. Mice were injected with ACTH and adrenals were collected at different timepoints after ACTH stimulation to assess the impact of ACTH on Pparg expression. Housekeeping gene: Gapdh. * <0.05 and FDR<0.05 (*).

Figure 6

Effect of ACTH treatment on MC2R expression in adrenocortical cell lines. Data are represented as mean ± SEM of log2Fold Change (log2FC) expression values normalized to the control samples. Housekeeping gene: ACTB. *p-value <0.05 and FDR<0.01.

Figure 7

Effect of rosiglitazone and ACTH treatment on cell viability in three different adrenocortical cell lines: NCI-H295R (A), MUC1 (B) and CU-ACC2 (C). Cell viability was evaluated by WST-1 assay (n=8 in each group). The cells were treated with increasing concentrations of rosiglitazone (0, 5, 10, 20 and 40 µM) and ACTH (0, 2.5,5,10 and 20 nm). The average absorbance values of each treatment group were normalized to background control group. Data are represented as percentage mean ± SEM of the normalized absorbance values. *p-value <0.0005 and FDR<0.01.

Figure 8

Effect of rosiglitazone and ACTH treatment on PPARG and MC2R expression in adrenocortical cell lines. The cells were treated with increasing concentrations of rosiglitazone (0,5,10,20 and 40 µM) and the expression was checked in the presence (2.5nm) and absence of ACTH. Data are represented as mean ± SEM of log2Fold Change (log2FC) expression values normalized to the control samples. Housekeeping gene: ACTB. *p-value <0.05 (*).

Figure 9

Effect of rosiglitazone (20µM) and ACTH (2.5 nm) treatment on the steroidome of adrenocortical cell lines. LC-MS/MS was used to quantify the steroidome in the supernatant of cells treated with rosiglitazone and ACTH and their respective controls. Importantly, the levels of glucocorticoids – cortisol (A), cortisone (B) and aldosterone (C), precursors of cortisol – 21-deoxycortisol [21-dF;(D)], 11-deoxycortisol [11-dF; (E)] and 17-hydroxyprogesterone [17OHP;(F)], androgens – testosterone (G), DHEA (H) and dihydrotestosterone [DHT;(I)] were quantified. Data are represented as mean ± SEM of individual concentration values (µg/L). *p-value <0.05 and FDR<0.05 (*).

Figure 10

Expression of PLIN1, PPARG target gene, in adrenocortical cell lines treated with rosiglitazone (20µM) and ACTH (2.5 nm). Data are represented as mean ± SEM of -dCT values. Housekeeping gene: ACTB. *represents p-value <0.05 and FDR<0.05 (*).