Transcription controls growth, cell kinetics and cholesterol supply to sustain ACTH responses

Abstract

Chronic ACTH exposure is associated with adrenal hypertrophy and steroidogenesis. The underlying molecular processes in mice have been analysed by microarray, histological and immunohistochemical techniques. Synacthen infused for 2 weeks markedly increased adrenal mass and plasma corticosterone levels. Microarray analysis found greater than 2-fold changes in expression of 928 genes (P < 0.001; 397 up, 531 down). These clustered in pathways involved in signalling, sterol/lipid metabolism, cell proliferation/hypertrophy and apoptosis. Signalling genes included some implicated in adrenal adenomas but also upregulated genes associated with cyclic AMP and downregulated genes associated with aldosterone synthesis. Sterol metabolism genes were those promoting cholesterol supply (Scarb1, Sqle, Apoa1) and disposal (Cyp27a1, Cyp7b1). Oil red O staining showed lipid depletion consistent with reduced expression of genes involved in lipid synthesis. Genes involved in steroidogenesis (Star, Cyp11a1, Cyp11b1) were modestly affected (P < 0.05; <1.3-fold). Increased Ki67, Ccna2, Ccnb2 and Tk1 expression complemented immunohistochemical evidence of a 3-fold change in cell proliferation. Growth arrest genes, Cdkn1a and Cdkn1c, which are known to be active in hypertrophied cells, were increased >4-fold and cross-sectional area of fasciculata cells was 2-fold greater. In contrast, genes associated with apoptosis (eg Casp12, Clu,) were downregulated and apoptotic cells (Tunel staining) were fewer (P < 0.001) and more widely distributed throughout the cortex. In summary, long-term steroidogenesis with ACTH excess is sustained by genes controlling cholesterol supply and adrenal mass. ACTH effects on adrenal morphology and genes controlling cell hypertrophy, proliferation and apoptosis suggest the involvement of different cell types and separate molecular pathways.

Keywords: ACTH; adrenal hyperplasia; adrenal hypertrophy; cholesterol.

Figure 1

ACTH infusion causes adrenal hypertrophy (A), increased corticosterone levels (B) and altered expression of gene transcripts (C and D). Values shown in A and B are mean values ± s.e.m., n = 6. Microarray values (grey bars) are compared with RT-PCR values (black bars) for a range of up and downregulated genes (D).

Figure 2

Heat maps showing expression genes involved in cell signalling that are upregulated (A) and downregulated (B) by ACTH. Each square represents gene expression of a single sample. Shades of blue and red indicate levels of expression below and above normalised values for individual genes.

Figure 3

ACTH infusion causes depletion of lipid droplets in adrenal cortex (A) and upregulation of gene transcripts associated with cholesterol biosynthesis (B), the cellular uptake of cholesterol (C) and the intracellular distribution of cholesterol (E). Downregulated transcripts associated with non-steroidogenic routes of cholesterol metabolism are shown (D). Values are means ± s.e.m. of n = 5 (control) and 6 (ACTH) adrenal glands.

Figure 4

ACTH infusion caused adrenocortical cell hypertrophy (A and B) and increased expression of transcripts associated with cell growth (C). (A) Shows representative H&E-stained sections with bar indicating magnification (100 µm). (B) Shows cross-sectional area of cells in zona glomerulosa (ZG), outer zona fasciculata (OZF), inner zona fasciculata (IZF) and medulla (MED) regions of the gland. Values are means ± s.e.m. of n = 6 (control) and 6 (ACTH) adrenal glands. (C) Shows increased expression of genes associated with cell size.

Figure 5

ACTH infusion increased adrenocortical cell proliferation (A and B) and the expression of gene transcripts associated with cell division (C). Sections of adrenal gland from control and ACTH were dual immunostained (A) for Ki67 (brown nuclei) and bromodeoxyuridine (purple nuclei) and analysed for numbers of positive nuclei (B). Values are means ± s.e.m. of n = 6 (control) and 6 (ACTH) adrenal glands; *P < 0.01, **P < 0.001). ACTH-induced fold changes in gene transcripts associated with cell proliferation are shown in (C).

Figure 6

ACTH treatment reduced Tunel staining in the adrenal cortex (A). Numbers of apoptotic cells corrected for cross-sectional area of cortex (B) were significantly reduced (P < 0.001). Values shown are means ± s.e.m. of n = 4 control and ACTH-treated adrenals. ACTH decreased expression (fold change) associated with apoptosis are shown in (C) (see also Supplementary Fig. 2).