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

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.


Introduction
Temporal control of adrenocortical responses to ACTH involves several processes, mediated by a common signalling system. In vivo and in vitro studies show that within five minutes, stress and ACTH cause increased adrenal corticosteroid release (Pearlmutter, et al. 1973;Walker, et al. 2015). Closer analysis of acute responses has demonstrated that activation of melanocortin receptors trigger cyclic AMP synthesis, leading to the synthesis of StaR which promotes the uptake of cholesterol into mitochondria (Artemenko, et al. 2001;Clark 2016;Simpson and Waterman 1983). Since steroid hormones are synthesised on demand, the availability of intramitochondrial cholesterol initiates steroidogenesis by providing substrate for the first rate-limiting enzyme in the steroidogenic pathway (Miller and Bose 2011). Longterm exposure to ACTH, requires changes in cholesterol supplysteroidogenic enzyme expression and adrenocortical cell hypertrophy and hyperplasia which take place over hours, days and weeks to maintain steroid output at a continuous high level. Although many of these key processes involve non-genomic enzyme activation, transcriptional control is also important.
Long term supply of cholesterol substrate is maintained by de novo synthesis, by uptake from the circulation and by release from stored intracellular lipid droplets. Key steps in de novo synthesis are hydroxymethyglutaryl CoA reductase (Hmgcr), squalene epoxidase (Sqle) and various post-lanosterol reductase and dehydrogenase steps (Ness 2015). Plasma LDL and HDL cholesterol (Brown, et al. 1979;Gwynne and Strauss 1982) are also available for steroidogenesis via LDL (Ldlr) and scavenger receptor (Scarb1) respectively. Within the adrenal cortex, cholesterol is stored as an ester in lipid droplets or utilized for steroidogenesis depending on the balance between lipase and esterifying enzyme activity (Beckett and Boyd 1977;Kraemer 2007;Thorngate, et al. 2002) Although prolonged stress or ACTH treatment causes adrenal gland hypertrophy, effects on the expression of genes encoding steroidogenic enzymes are less profound (Lehoux, et al. 1998;Ye, et al. 2008). In fact, it may be that induction of intra-adrenal steroid hormone metabolising enzymes help mitigate the effects of excess ACTH. In sheep, for example, there is a marked increase in 20 hydroxylation of corticosteroid intermediates which have no clearly defined biological activity (Butkus, et al. 1985). Similarly, there are reports that adrenal 5α reductase and sulfotransferase activities may affect the secretion of biologically active hormones (Al-Dujaili, et al. 2011;Luu-The, et al. 2005).
Genomic and somatic mutations of various genes have been identified that explain excess steroid production in cortisol and aldosterone producing adenomas (Calebiro, et al. 2015;Zennaro, et al. 2015). These involve gain/loss of function that affect adrenocortical signalling processes. Although these genes are required, physiological control of steroidogenesis is not necessarily mediated by regulation of their expression. Moreover, there is a need to determine that signalling elements responsible for acute changes in steroid output are the same as those mediating adrenocortical adaptation to chronic stress or prolonged ACTH exposure.
In this study, we have used microarray analysis of mouse adrenal tissue to gain a comprehensive picture of transcriptional control processes affecting cell signalling, cholesterol supply and cell turnover in response to chronic stimulation of corticosterone synthesis in mice infused with an ACTH analogue, Synacthen. We found modest changes in genes encoding steroidogenic enzymes. Our data suggests that enhanced steroidogenic capacity reflects increases in cell size and a shift in the balance between proliferation and apoptosis that increases cell number. Sterol/lipid metabolic pathways are also changed in several ways to allow cholesterol to be channelled towards steroidogenesis.

Materials & Methods
All experiments involving animals were approved by the University of Edinburgh Animal Welfare and Ethical Review Body and were carried out in strict accord with accepted standards of humane animal care under the auspices of the Animal (Scientific Procedures) Act UK 1986. Groups (n =5/6) of age-matched male C57BL6 mice (Harlan Olac) weighing approximately 25g were fed a diet containing 0.3% Na (SDS Diets, Witham, Essex, UK) with free access to water in a temperature and light-controlled (12h light/dark cycle; 07.00h lights on) room. Mice were infused sc via miniosmotic infusion pumps (Alzet Cupertino, Model 2002) with either vehicle (0.154M NaCl) or ACTH (Synacthen Ciba-Geigy, UK; 3µg/d). At the end of the study mice were killed by carbon dioxide at the nadir of the circadian cycle.
Pairs of adrenal glands were collected into a solution of RNeasy (Qiagen Ltd, Crawley, UK).
Each adrenal was carefully trimmed free of fat under a dissecting microscope and weighed.

Histology and immunohistochemistry.
Additional groups of mice were infused with ACTH or saline as above to assess effects on adrenal morphology and cell proliferation. Blood was collected by cardiac puncture into lithium heparin tubes for corticosterone measurements by ELISA (Al-Dujaili, et al. 2009). In one experimental cohort, bromodeoxyuridine (1mg/ml) was added to the minipump infusates to monitor cell proliferation. After a two-week infusion, mice were killed by decapitation and tissues collected for fixation in buffered formalin and embedded in paraffin wax. BrdU positive and Ki67 positive nuclei in mid-adrenal sections were located by immunohistochemistry as previously described (Chang, et al. 2013). Cells with Ki67 positive nuclei were counted in the zona glomerulosa/outer zona fasciculata region of the cortex and numbers were normalised to length of capsule perimeter. Haematoxylin and eosin stained sections were used to estimate cell size in different regions of the adrenal cortex (Chang et al.

2013).
To assess the cellular storage of cholesterol, adrenals were collected without fixation and stored at -80C before cryosectioning for oil red O staining of lipid droplets. Images were captured with a Niko Coolpix color camera with a Zeiss Axioskop 2 compound microscope and MCID imaging software (Imaging Research Inc, St Catharines, Ontario, Canada) Apoptosis was analysed using a Roche Tunel staining kit (Sigma-Aldrich, UK).

Microarray processing
RNA for microarray analysis was prepared from individual adrenal glands (n = 5 and 6 for saline and ACTH-treated mice respectively) using TRIzol® (ThermoFisher Scientific, Loughborough, UK) and then processed through standard Affymetrix protocols, with one  Table 1.

Real time RTPCR
Based on initial findings from microarrays, selected genes were quantified by pre-optimised RT-PCR assays in RNA from individual adrenals of separate cohorts of saline and ACTH-treated mice that were killed with CO 2 Total RNA was extracted from tissue samples using the Qiagen RNeasy system and reverse transcribed into cDNA with random primers using the QuantiTect DNase / reverse transcription kit (Qiagen Ltd). cDNA (equivalent to 1ng total RNA) was incubated in triplicate with gene specific primers and fluorescent probes (using pre-designed assays from Applied Biosystems, Warrington, UK) in 1x Roche LightCyclerR480 Probes mastermix. PCR cycling and detection of fluorescent signal was carried out using a Roche LightCyclerR480. A standard curve was constructed for each primer probe set using a serial dilution of cDNA pooled from all samples. Results were corrected for the mean of expression of beta-actin and 18S ribosomal RNA. Neither 18S RNA nor beta actin were affected by ACTH treatment.

Statistics
Data are presented as mean ± SE. After tests for Gaussian distribution, comparisons were made using either unpaired t-test or ANOVA with Bonferroni post hoc testing; P ≤ 0.05 were considered statistically significant. Gene expression profiles with Pearson correlation coefficients ≥ 0.9 were analysed for clustering.

Adrenal response to ACTH treatment
Mice infused with ACTH had larger adrenals and higher plasma corticosterone levels ( Figure   1A & B). Previous studies of urinary corticosterone excretion in this model indicate that ACTH treatment produces a sustained increase in steroidogenesis (Dunbar et al. 2010). Body weight gain was not affected. After normalisation and correction for multiple testing, microarray analysis indicated that ACTH significantly affected the expression of approximately 9000 gene transcripts (P <0.05; figure 1C); 397 and 531 annotated genes were up-and down-regulated respectively by ≥ two-fold (P < 0.001). To validate the microarray results, a parallel analysis was carried out using PCR methods to quantify mRNA of representative genes that were up-and down-regulated in the microarray. The choice of genes reflected a range of responses and their possible involvement in processes controlling signalling and sterol metabolism (Lpl,Srd5a1,Scarb1,Ren1,Mrap), cell proliferation (Fgfrl1, Impdh1) and apoptosis (Cidea,Casp12,Clu,Trib3,Elmo1). The patterns of change for the selected genes were broadly similar for microarray and qPCR methods ( Figure 1D). It is notable, that genes encoding enzymes that metabolise steroids were also affected.

Adrenal Size
Cell Hypertrophy: Part of the ACTH-induced increase in adrenal mass is due to adrenocortical cell hypertrophy. The cross-sectional area of zona fasciculata cells, the main cell type of the cortex, was >2 fold greater but cells of other cortical zones were also Immunohistochemistry identified approximately ten times more BrdU-positive than Ki67positive cells ( Figure 5B). Interestingly, ACTH increased the numbers of BrdU-and Ki67positive cells to a similar degree indicating that proliferative effects were sustained throughout the period of treatment. Increases in cell proliferation were matched by expression of genes associated with various aspects of cell division ( Figure 5C).

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Apoptosis: The net effect of ACTH on adrenocortical volume may involve a decrease in cell death as well as increases in cell hyperplasia and hypertrophy ( figure 6A & B). Figure  Adrenal cholesterol is also provided by circulating lipoproteins: predominantly LDL in humans via LDL receptors (Ldlr) and HDL in rodents via scavenger receptors (Scarb1) (Gwynne and Strauss 1982;Kraemer 2007;Rigotti, et al. 1996). ACTH increased expression of both Ldlr and Scarb1 as well as Ldlrap1 which facilitates LDL uptake. Apoe, which is also known to affect LDL receptor activity and is highly expressed specifically in the adrenal cortex, was not affected. This contrasts with previous reports on apolipoprotein E protein and mRNA levels (Cheng, et al. 1998;Prack, et al. 1991) . However, the gene Lrp8 which encodes a receptor for ApoE was upregulated as was Vldlr; both genes have been associated with the reelin signalling system which controls neural development and plasticity (Herz and Chen 2006) suggesting an alternative adrenal function. Similarly, Hdlbp, although increased by ACTH treatment and with an affinity for HDL (Fidge 1999), is also known as vigilin, an RNA binding protein with wider functions that might be independent of steroidogenesis (Mobin, et al. 2016) .
Cholesterol from circulating lipoproteins is taken up in the form of esters requiring hydrolysis to render free cholesterol for steroidogenesis. Two genes encoding adrenal cholesterol esterases have been identified: hormone sensitive lipase (Lipe) (Kraemer 2007) and more recently neutral cholesterol ester hydrolase (Nceh1) (Ohta, et al. 2011). Control of hormone sensitive lipase activity may be post-translational (Yeaman 2004); Lipe expression is not significantly affected in the present study (-1.67, P = 0.1). In contrast, Nceh1 is modestly increased by ACTH treatment (1.82, P<0.0001). It is notable that genes encoding factors required for intracellular cholesterol trafficking (Npc1, Stx11) (Li, et al. 2016;Lin, et al. 2016) were also up-regulated.
Under normal conditions, excess cholesterol is stored as esters in lipid droplets. As evidenced previously (Cheng et al. 1998;Thorngate et al. 2002) and here by oil red O staining, the zona fasciculata cells of ACTH-treated mice are deplete of lipid. A pattern of reduced expression of genes involved in cholesterol and lipid metabolism is consistent with this depletion. Plin4 (perilipin 4, a component of the droplet coat) (Kraemer, et al. 2013) is down-regulated as are Acat1 and 2, the transferases that esterify cholesterol. However, these transferases are also known to be allosterically activated by sterols (Rogers, et al. 2015). In addition, many genes implicated in the biosynthesis of the lipids which are co-substrates in cholesterol ester synthesis are down-regulated, implying that triglycerides are normally produced locally to maintain cholesterol ester reserves (Supplementary Figure 2).
A further way of optimising cholesterol supply is to limit non-steroidogenic routes of metabolism. Excretion of cholesterol is via hepatic bile acid synthesis and although the adrenal does not produce significant amounts of bile, genes encoding three genes in bile synthesis are down-regulated as is Fxr, a key transcription factor regulating the bile pathway (Russell 2009). Stard10 which binds phosphatidyl choline and is involved in bile acid metabolism, is also down-regulated (Alpy and Tomasetto 2014). The work of Schroeder et al (Schroeder, et al. 2010) has implicated sterol carrier protein-2 (Scp2) and caveolin-1 (Cav1) as factors in liver controlling the intracellular distribution, efflux and esterification of cholesterol. It is significant therefore that ACTH downregulated both Scp2 (-1.62 fold, P <0.001) and Cav1 (-2.37 fold, P <0.0001) expression.
The first step in steroidogenesis is the mitochondrial side chain cleavage of cholesterol to produce pregnenolone. Cleavage activity is determined by cholesterol uptake across the mitochondrial membrane. Papadopoulos and colleagues have suggested that this involves a complex of five proteins termed a transduceosome (Liu, et al. 2006). Although this hypothesis is controversial (Selvaraj, et al. 2016), of the genes encoding these five proteins, Vdac1 and Prkar1a are upregulated two fold (P<0.001), Star and Acbd3 are slightly increased (1.2 fold, P<0.01) and Tspo (peripheral benzodiazepine receptor) expression is unaffected.
The relative change in genes encoding steroidogenic enzymes is modest (<1.3 fold) reaffirming studies that show cholesterol supply is critical in maintaining high corticosteroid output when enzyme expression is non-limiting. However, it is notable that ACTH has profound effects on other genes affecting steroid metabolism. Akr1c18, which encodes a 20α hydroxysteroid dehydrogenase enzyme, was switched on in ACTH-treated adrenals (>80 fold). This is consistent with observations in sheep where circulating levels of dihydroxyprogesterone were increased in ACTH-induced hypertension (Butkus et al. 1985).
Previous studies in mouse indicate that adrenal Akrc18 expression is normally high in prepubertal and low in adult males (Hershkovitz, et al. 2007). In contrast expression of Sd5a2 that encode a 5α steroid reductase, which is normally high in the male mouse adrenal gland (Luu-The et al. 2005), was decreased more than 5-fold by ACTH. In general, the activity of these enzymes controls the availability of intermediates in the corticosteroid pathway thereby affecting throughput to biologically active hormonal end products.
A major contribution to steroidogenic capacity is the marked threefold increase in adrenal mass. The morphology of adrenals from ACTH-treated mice indicate this is due to expansion of the cortex rather than the medulla and is a function of both the size and numbers of parenchymal cells. Histology shows that cell hypertrophy is predominantly a feature of the zona fasciculata. The twofold increase in cross-sectional area with ACTH treatment is perhaps an underestimate since volume expansion is offset by depletion of lipid droplets.
Increased expression of Cdkn1a, a marker of cell cycle arrest, suggested cell cycle progression beyond the G1 stage is inhibited in some cells. The close correlation of Cdkn1a expression with other genes (correlation coefficient >0.9, P<0.0001) reflects changes in growth and de novo protein. Cdkn1c, a gene known to affect adrenal cell turnover in embryonic life (Nagahama, et al. 2001), was also increased (4.12 fold) but with a pattern of change that did not strongly correlate with that of Cdkn1a (r = 0.62).
BrdU and Ki67 immunostaining demonstrated ACTH caused a sustained stimulation of cell division which appeared to be initiated in a subcapsular region of the gland with displacement of newly divided cells inwards (Chang et al. 2013). The majority of hypertrophied cells throughout the cortex were not immunostained suggesting that increased cell size is independent of mitosis. In line with Ki67 staining, expression of Mki67 was also increased as were a number of genes linked to cell proliferation and the cell cycle. Tk1, encoding thymidine kinase, another established marker of proliferation, was increased 2.25 fold (P < 0.0001). As with genes controlling cell hypertrophy, expression of genes associated with proliferation were closely clustered. Cell number is determined by cell death (apoptosis) as well as division. The term apoptosis was first used in the seminal work of Kerr et al (Kerr, et al. 1972) to describe programmed cell death in several experimental models including the effects of ACTH withdrawal on the rat adrenal cortex. Not surprisingly, ACTH infusion in the present experiment had anti-apoptotic effects which were linked to decreased expression of genes like Casp12. These genes include histocompatibility factors, which previous studies have shown are expressed in inner regions of the cortex with a role in apoptosis (Wolkersdorfer, et al. 1996). Similarly, various components of complement activation that have been shown to play a role in the clearance of apoptotic cells (albeit in non-adrenal cells (Ogden and Elkon 2006)) were decreased. It should be stressed, however, that the level of apoptosis in a normal gland is low so that an ACTH-induced reduction in apoptosis may be difficult to detect, particularly if cell clearance is efficient (Carsia, et al. 1996). Nevertheless, Tunel staining in the present study showed a fivefold decrease with ACTH treatment. This is perhaps an overestimate given that the number of apoptotic cells is expressed relative to cross sectional area of the cortex and the cross-sectional area of cells from ACTH-treated adrenals is up to two times greater than controls.
The diversity of cellular effects underlying the response to chronic ACTH treatment is reflected in the range of genes involved in signalling. ACTH receptor regulation of adrenal activity is mediated through the cyclic AMP cascade (Gallo-Payet 2016) but few of the genes involved are affected. Adenylate cyclase isoforms are generally down-regulated and Pde8b, which is negatively associated with steroidogenic activity (Horvath, et al. 2008), was upregulated by ACTH treatment. Genes involved in protein kinase A activity which are mutated in some patients with ACTH-independent Cushings (Calebiro et al. 2015) were up-regulated as was the protein kinase A anchor protein Akap2. The upregulated cluster was enriched with genes implicated in processes downstream of the cyclic AMP cascade including genes for transcription factors (Nr5a1, Creb3l2), for cell proliferation and hypertrophy (Rras2,Cdkn1a,Rrcc2,Igflr,Shmt1) and for snare proteins (Stx11). Genes like Prkcd, Srxn1 and Inha are known to be involved in steroidogenic control (Brizuela, et al. 2006;Hofland and de Jong 2012;Kil, et al. 2012) whereas the function of others (Gucalb, Fam161a) have yet to be defined.
In summary, a large number of adrenal genes are differentially affected by chronic ACTH treatment. Upregulated genes include those implicated in cell division and cell hypertrophy as well as those promoting cholesterol supply from de novo synthesis and uptake from the circulation. Downregulated genes are those involved in apoptosis, intracellular storage of cholesterol esters and non-steroidogenic metabolism of cholesterol. Preliminary observations indicate that genes controlling each of these different processes are co-ordinately regulated.
Theoretically, this information could be used to target genes to manipulate glucocorticoid output or ACTH responsiveness. For example, the adverse effects of glucocorticoid hormones could be ameliorated in patients with Cushing's disease due to excess pituitary ACTH or Cushing's Syndrome caused by ectopic ACTH production, It is important, however, to reiterate that many genes that are transcriptionally regulated by ACTH encode factors which are subject to non-genomic control. Finally, excessively high glucocorticoid hormone levels may, secondarily, affect gene expression in the zona glomerulosa and the adrenal medulla with consequences for aldosterone and catecholamine synthesis.     Values are means ± SEM of n =5 (control) and 6 (ACTH) adrenal glands.   that are associated with cell hyperplasia, hypertrophy and apoptosis. Lines represent Pearson correlation coefficients between genes that are ≥ 0.9. The cluster on the left comprises upregulated genes that are associated with cell hyperplasia and hypertrophy: note the genes in grey include Mki67, Tk1 and Cdc2a that are involved in cell proliferation. The genes in yellow on the right are down-regulated genes including Casp12 and various histocompatibility and complement system genes that are associated with apoptosis.