ACTH is a potent regulator of gene expression in human adrenal cells

Abstract

The adrenal glands are the primary source of minerocorticoids, glucocorticoids, and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by ACTH. Although it has been established that ACTH can stimulate steroidogenesis, the effects of ACTH on overall gene expression in human adrenal cells have not been established. In this study, we defined the effects of chronic ACTH treatment on global gene expression in primary cultures of both adult adrenal (AA) and fetal adrenal (FA) cells. Microarray analysis indicated that 48 h of ACTH treatment caused 30 AA genes and 84 FA genes to increase by greater than fourfold, with 20 genes common in both cell cultures. Among these genes were six encoding enzymes involved in steroid biosynthesis, the ACTH receptor and its accessory protein, melanocortin 2 receptor accessory protein (ACTH receptor accessory protein). Real-time quantitative PCR confirmed the eight most upregulated and one downregulated common genes between two cell types. These data provide a group of ACTH-regulated genes including many that have not been previously studied with regard to adrenal function. These genes represent candidates for regulation of adrenal differentiation and steroid hormone biosynthesis.

Conflict of interest statement

The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

Figures

Fig.1. Time-dependent effects of ACTH on cortisol production in human AA cells

Primary human AA cells were prepared as described under Methods, and plated at the density of 200,000 cells per well in 24 well dishes. Cells were treated with ACTH (10 nM) for the indicated times and cortisol was quantified in the medium using EIA. Cortisol data was normalized to protein per well and expressed as the fold change over basal. Results represent the mean ±S.E.M of data from at least three independent experiments. Statistics were calculated using one way ANOVA followed by Dunnett test. *, P<0.05, **, P<0.01.

Fig.2. Microarray analysis of ACTH effects on AA cell gene expression

Panel A. Scatter plot comparing gene expression between basal and ACTH treated samples. Total RNA from three sets of primary adrenal culture samples treated with/without ACTH were used for six oligonucleotide microarrays and the data were combined to form this plot. The graph represents 37,846 transcripts that were found to have a signal which indicated that it was present in at least one tissue sample. The transcripts with the highest variation between the basal and ACTH are labeled. Panel B. Heatmap of the fifteen most differentially expressed transcripts between three basal and three ACTH treated samples. The top ten upregulated and top five downregulated genes are given here. Colors represent the expression level from the median of all the samples for each probe set. Results represent data from three experiments using cells isolated from three independent donor adrenal glands.

Fig.3. Time-dependent effects of ACTH on cortisol and DHEA-S production in FA primary cultures

Primary human FA cell were prepared as described in Method section, and plated at a density of 300,000 cells per well in 24-well dishes. The day before experiments, cells were changed to 1% low serum medium overnight. Cells were treated with ACTH (10 nM) for the indicated times followed by quantification of medium cortisol and DHEA-S using EIA. Steroid data was normalized to protein per well and expressed as fold change over basal. Results represent the mean ±S.E.M of data from at least three independent experiments. Statistics were calculated using one way ANOVA followed by Dunnett test. *, P<0.05, **, P<0.01.

Fig.4. Microarray analysis of ACTH effects on FA cell gene expression

Panel A. Scatter plot comparing gene expression between basal and ACTH treated samples in FA cells. Total RNA from three sets of primary adrenal samples treated with/without ACTH were used for oligonucleotide microarray analysis and the data were combined. The graph represents 18,391 transcripts that were found to have a signal which indicated that it was present in at least one cell sample. The transcripts with the highest variation between the basal and ACTH are labeled. Results represent data from three experiments using cells isolated from three independent adrenal glands. Panel B. Heatmap of the fifteen most differentially expressed transcripts between basal and ACTH treatment. The top ten upregulated and top five downregulated genes are shown. Colors represent the expression level from the median of all the samples for each probe set. Results represent data from three experiments using cells isolated from three independent FA glands.

Fig.5. Common genes that are increased by ACTH treatment in AA and FA cells

Panel A. Venn diagram of genes upregulated by ACTH by at least 4 fold in AA and FA cells. Grey: AA cell. White: FA cells. Panel B. Heatmap of the twenty commonly regulated genes between AA and FA after ACTH treatment. Colors represent the expression level from the median of all the samples for each probe set. Results represent data from three experiments using cells isolated from three independent donor adrenal glands.