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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-1309
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 12 6638-6649
Copyright © 2005 by The Endocrine Society

Steroidogenesis in Aldosterone-Producing Adenoma Revisited by Transcriptome Analysis

Guillaume Assié, Colette Auzan, Jean-Marie Gasc, Erno Baviera, André Balaton, Jean-Marc Elalouf, Xavier Jeunemaitre, Pierre-François Plouin, Pierre Corvol and Eric Clauser

Institut National de la Santé et de la Recherche Médicale, Unité 567, Centre National de la Recherche Scientifique 8104, University Paris 5, Cochin Institute (G.A., C.A., E.C.), 75014 Paris, France; Institut National de la Santé et de la Recherche Médicale, Unité 36, Collège de France (J.-M.G., X.J., P.C.), 75005 Paris, France; Department of Pathology, Saint Joseph Hospital (E.B., A.B.), 75014 Paris, France; Department of Cellular Biology, Commissariat à l’Energie Atomique (J.-M.E.), 91400 Saclay, France; and Department of Vascular Medicine and Arterial Hypertension, Georges Pompidou European Hospital (P.-F.P.), 75015 Paris, France

Address all correspondence and requests for reprints to: Dr. Eric Clauser, Institut National de la Santé et de la Recherche Médicale, Unité 567, Centre National de la Recherche Scientifique 8104, Université Paris 5, Endocrinology Department, Institut Cochin, 24 rue du Fg Saint Jacques, 75014 Paris, France. E-mail: clauser{at}cochin.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Primary aldosteronism (PAL) is the most frequent cause of secondary arterial hypertension. In PAL, aldosterone production is chronic, excessive, and autonomous.

Objective: The objective of this study was to identify the angiotensin-II independent alterations of steroidogenesis responsible for PAL.

Design: Genomewide gene expression was compared in two tissues differentiated for aldosterone production, both nonstimulated by circulating angiotensin II and differing in their autonomy to produce aldosterone: aldosterone-producing adenoma (APA) and its adjacent dissected zona glomerulosa (ZG).

Setting: The setting of this study was the Comete Network.

Patients: Patients with APA were studied.

Intervention: Transcriptome comparison was made of one APA and its adjacent ZG by serial analysis of gene expression; validation by in situ hybridization was performed for 19 genes in 11 samples.

Outcome: The study outcome was genes differentially expressed in APA and adjacent ZG.

Results: Activation of steroidogenesis in PAL is restricted to the overexpression of the enzymes producing aldosterone-specific steroids, aldosterone synthase and also 21-hydroxylase, suggesting that upstream precursor production is not limiting. Increased expression of high-density lipoprotein receptor, adrenodoxin and P450 oxidoreductase suggests that these systems provide cholesterol and electrons to the mitochondrial steroidogenic enzymes. As for acute stimulation of aldosterone production, an activation of calcium signaling is suggested by concordant overexpression of calcium-binding proteins or effectors. Calcium activation may result from an abnormal activity of Gq protein-coupled receptors. This calcium activation may be the starting point of the other gene expression changes observed in APA. Finally, other differentially expressed genes include three genes encoding unidentified proteins.

Conclusion: This work provides an original and integrated view of the mechanisms of aldosterone production in PAL.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE RENIN-ANGIOTENSIN-aldosterone system is a major endocrine system regulating blood pressure and hydroelectrolytic balance. Aldosterone is a steroid hormone produced by the zona glomerulosa (ZG), the most superficial region of the adrenal cortex. Its main physiological actions are sodium reabsorption and potassium secretion in the renal distal tubule. Primary aldosteronism (PAL) is characterized by chronic, excessive, autonomous secretion of aldosterone by the adrenal gland. This overproduction of aldosterone leads to hypertension, accounting for up to 10% of hypertensive patients (1), and cardiovascular fibrosis (2). The pathophysiology of PAL has been investigated in both sporadic and familial forms of the disease. However, the causal determinants and cellular changes responsible for aldosterone production in PAL are still poorly understood.

Rare familial forms of PAL have been described. Familial hyperaldosteronism type 1 is due to an unequal crossing over between two steroidogenic enzyme genes (3). This results in excessive and ectopic production of aldosterone synthase (CYP11B2), which can be blocked by glucocorticoids (4).

Sporadic PAL results from two major types of adrenal lesion: 1) aldosterone-producing adenoma (APA) is a benign tumor of the adrenal cortex in which the adjacent and contralateral ZG is nonfunctional, due to a negative feedback loop of the renin-angiotensin-aldosterone system (5); and 2) bilateral adrenal hyperplasia involves the entire ZG of both adrenal glands (6).

APA has been the principal model used to study changes in gene expression in sporadic PAL. Numerous genes, such as aldosterone synthase (7, 8, 9), have been identified as differentially expressed in APA and adrenal cortex. However, these studies are limited to a candidate gene approach and use in general as control tissue the entire adrenal cortex, which mostly produces cortisol.

The changes in gene expression responsible for aldosterone production may also be investigated in animal and cell culture models after acute stimulation. Studies have identified aldosterone synthase expression (10), cholesterol supply to mitochondria (11), and calcium signaling (12) as major regulatory targets for aldosterone production. However, these models are limited for the investigation of PAL pathophysiology, because they explore in vitro acute and stimulated aldosterone production.

To overcome all these limitations and problems, we designed an original study comparing gene expression in an autonomous aldosterone-producing tissue, the APA, and in a reference tissue also differentiated for aldosterone production, but that does not produce aldosterone in the absence of circulating angiotensin II, the adjacent ZG. Transcriptome comparison is based on serial analysis of gene expression (SAGE; SAGE data are available at GEO www.ncbi.nlm.nih.gov/geo/ under accession no. GSM36126 and GSM36127, corresponding to the crude SAGE data for the APA and its adjacent ZG) (13). This study is the first integrated and exhaustive analysis of APA transcriptome, which sets up the bases of the mechanisms of the disease.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects and adrenal samples

All samples were obtained from patients undergoing surgery for APA (Table 1Go). Patients were treated and underwent surgery at a single referent medical center. The reality of APA diagnosis is supported by the selective adrenal venous sampling when available and for all 11 patients, by normal postoperative values of serum potassium, plasma active renin concentration, plasma aldosterone concentration, and urinary aldosterone secretion. In addition, for almost all patients, in situ hybridization for aldosterone synthase or 21-hydroxylase shows an extensive labeling of the adenoma, without any signal in the adjacent zona glomerulosa, excluding hyperplasia. Therefore, postoperative hypertension observed in some patients is linked to nonaldosterone-dependent mechanisms. Patients gave informed, written consent, and samples were taken within the Comete network, with the approval of the local ethics committee.


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  		TABLE 1. Baseline characteristics and postoperative outcome in patients with APA

 
The pathological adrenal gland was collected during surgery and immediately examined by the pathologist. After macroscopic examination, adrenal glands with a single, well-delimited nodule were further dissected for study. A 5-mm3 fragment was cut out of the center of the adenoma and immediately frozen in liquid nitrogen for RNA preparation. We removed the fat from the surrounding nontumoral adrenal gland and pulled the capsule out with a pair of forceps, a method adapted from the classical handling to separate the ZG from other adrenal cortex layer in rats (14). Fragments of ZG (5 mg) were either frozen in liquid nitrogen for RNA preparation or fixed in paraformaldehyde, dehydrated, and embedded in paraffin for standard histological examination and in situ hybridization (ISH).

Construction and analysis of SAGE libraries

SAGE libraries were prepared from an APA and the adjacent nontumoral ZG tissue from the same patient. Given the limited amounts of nontumoral tissue available, we used a modified version of the SAGE method (13) known as SADE (15). Polyadenylated RNA was purified from total RNA using thymidine phosphate polymer-anchored magnetic beads (Dynal Biotech, Great Neck, NY). Specific tags were isolated from cDNAs using Sau3A I (New England Biolabs, Beverley, MA) as the anchoring enzyme. These specific tags were ligated together to form concatemers, amplified, subcloned, and sequenced according to standard procedures. Sequence data were analyzed using the SAGE software 3.04 (www.sagenet.org), which first extracts, classifies, and counts the tags from the sequences and then compares the two libraries by means of tests based on Monte Carlo simulations. Each tag was identified with the SAGEmap resource (www.ncbi.nlm.nih.gov/SAGE/), and identification was confirmed manually on the basis of strict criteria: 1) unique matching to a nuclear genomic transcript identified in the EMBL, DDBJ, or GenBank mRNA databases, using the BLASTN algorithm (www.ncbi.nlm.nih.gov/BLAST/); 2) correct position and orientation in the transcript (immediately downstream from the most 3' GATC anchoring site); 3) not differing from a more abundant tag by a single nucleotide; and 4) no match with Alu sequences or the linker sequence used for SAGE library construction. Tags matching mitochondrial DNA were referred to as mitochondrial tags.

ISH

ISH was performed on tissue sections containing both the APA and the adjacent ZG, which were therefore hybridized under identical conditions. cDNAs were obtained by RT-PCR from APA RNA and were subcloned into the pGMTeasy plasmid (Promega Corp., Madison, WI). The specific probes for the various genes are summarized in Table 2Go. For CYP11B2 and CYP11B1, which have 93% identical sequences, we used synthetic oligonucleotide probes, as described by Pascoe et al. (16). Labeled sense and antisense riboprobes were transcribed in vitro from cloned cDNA using the Sp6 and T7 RNA polymerases (Roche, Indianapolis, IN) in the presence of [35S]UTP (Amersham Biosciences, Arlington Heights, IL). ISH was carried out as previously described (17).


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  		TABLE 2. Specific sequences used for in situ hybridization

 
We carried out a semiquantitative comparison of gene expression between APA and adjacent ZG cells, using different samples and two blind observations. A black and white dark-field image of APA and of the adjacent ZG at a magnification of x25 was captured using a digital camera (Coolsnap, model DMC1, Polaroid, Cambridge, MA) with constant parameters. The images were juxtaposed, using Adobe Photoshop 5.5 software (Adobe Systems, Inc., San Jose, CA) for display. Differences in labeling were assigned to seven categories as follows: 1) strong labeling in APA and no labeling in the adjacent ZG; 2) labeling in APA at least twice as strong as that in the adjacent ZG; 3) labeling more intense in APA than in ZG, but by a factor of less than 2; 4) no difference; 5) labeling more intense in ZG than in APA, but by a factor of less than 2; 6) labeling in ZG at least twice as strong as that in APA; and 7) strong labeling in ZG and no labeling in the adjacent APA.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Gene expression profiles in APA and adjacent ZG

We compared the transcriptomes of one APA and the adjacent ZG from the same adrenal gland. The clinical characteristics of the patient are summarized in Table 1Go (patient 11). Macroscopic and histological examination identified a single, 10-mm, yellow tumor without surrounding adrenal hyperplasia. ISH of this adrenal cortex specimen using an aldosterone synthase (CYP11B2) probe resulted in strong, homogeneous labeling of the APA cells, whereas labeling of the adjacent ZG cells was very weak, confirming the negative retrocontrol (Fig. 1Go, A–C). Histological examination of the ZG control sample indicated that the vast majority of cells were ZG cells surrounded by a capsule with very few fibroblasts and some fasciculata cells (Fig. 1DGo).



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  		FIG. 1. Morphology and ISH with an aldosterone synthase probe of the adrenal samples used to generate SAGE libraries. A, Histological section of a single APA with the adjacent adrenal cortex from patient 11 after toluidine blue staining (lower part). Dark-field photomicrograph of an adjacent part of the section showing the location of aldosterone synthase (CYP11B2) as detected by ISH in the APA and not in the adjacent ZG (upper part). B and C, Higher magnification (bright-field) after ISH, using the same CYP11B2 probe for APA (B) and adjacent ZG (C) from the same adrenal gland. D, Histology of the ZG sample obtained by decapsulation and used for the construction of the SAGE library (from patient 11). ZF, Zona fasciculata; C, capsule. Scale bar, 50 µm (A and D) and 10 µm (B and C).

 
Two SAGE libraries were generated from APA and adjacent nontumoral ZG mRNA; 20,371 (APA) and 18,686 (ZG) mRNA tags were obtained. The number of tags recorded at least twice was 1,916 in the APA library and 2,073 in the ZG library. Of the 5,754 and 5,239 tags present as single copies in the APA and ZG libraries, respectively, 712 were common to both libraries. The complete datasets for these libraries are available at the GEO site (www.ncbi.nlm.nih.gov/geo/; accession no. GSM36126 and GSM36127).

Comparison of the two libraries showed that 260 tags were differentially expressed in the APA and adjacent ZG (P < 0.05, in Monte Carlo simulation analysis). These differentially expressed tags were classified according to expression ratio between the two tissues. The 50 tags most strongly overexpressed in the APA (Table 3AGo) were 3–40 times more abundant in the APA. Sixteen of these 50 tags corresponded to mitochondrial transcripts or nuclear transcripts encoding mitochondrial proteins, suggesting that mitochondrial activity increases in APA. Nineteen correspond to well-identified nonmitochondrial transcripts (Table 3AGo), and 15 could not be reliably identified (see Subjects and Methods). The 50 tags most strongly underexpressed in the APA were less abundant in this tissue by a factor of 5–25. Thirty-eight of these tags correspond to well-identified nonmitochondrial transcripts (Table 3BGo). Interestingly, seven of these transcripts encoded ribosomal proteins or translational regulatory proteins, suggesting potential differences in the overall rate of protein synthesis between the two tissues.


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  		TABLE 3. The genes displaying the most differential expression between APA and adjacent ZG

 
For 19 candidate genes identified by SAGE as differentially expressed between APA and the adjacent ZG, we carried out ISH on adrenal tissues from 10 patients with APA, whose clinical characteristics are summarized in Table 1Go.

Specific modulation of the steroidogenic pathway

Cholesterol yields aldosterone through a series of steroidogenic enzyme reactions (Fig. 2aGo). Beside aldosterone synthase (CYP11B2) (10), little is known about the expression of genes encoding other steroidogenic enzymes in PAL patients. We analyzed the differential expression of these genes.



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  		FIG. 2. Differential expression of genes encoding steroidogenic enzymes. a, Schematic representation of mineralocorticoid steroidogenesis. In black, genes expressed more strongly in the APA than in the adjacent ZG; in gray, genes with no differential expression. HSD3B2, 3ß-Hydroxysteroid dehydrogenase 2; CYP11A1, cholesterol side chain cleavage enzyme; CYP11B2, aldosterone synthase; CYP21A2, 21-hydroxylase. b, Differential expression of steroidogenic enzymes estimated by SAGE and ISH. Abundance of the SAGE tags is expressed as the number of copies for a total of 20,371 tags in the APA library and 18,686 in the ZG library. The P values were determined according to Monte Carlo simulations. The samples used for the SAGE experiments were taken from patient 11. ISH was performed on several samples (from patients 1–11). For each sample, differential expression was semiquantified, classified as follows: +++, expressed exclusively in APA; ++, more strongly expressed in APA (by a factor >2); +, more strongly expressed in APA (by a factor <2); =, equally strongly expressed in APA and ZG; –, more strongly expressed in ZG (by a factor <2); ––, more strongly expressed in ZG (by a factor >2); –––, expressed exclusively in ZG. The P values were determined by the Wilcoxon rank test (two-sided). c, An APA (A and C) and its adjacent adrenal cortex (B) or ZG (D) from patient 3 after ISH using a 21-hydroxylase (CYP21A2) probe. A and B, Bright-field and adjacent dark-field photomicrographs showing strong labeling of the adenoma (A) and zona fasciculata (B), but not of the adjacent ZG (B). C and D, Higher magnification (bright-field) of the same APA and adjacent ZG, confirming differential expression. ZF, Zona fasciculata; C, capsule. Scale bar, 50 µm (A and B) and 10 µm (C and D).

 
The production of larger amounts of aldosterone synthase mRNA in the APA than in the adjacent ZG was confirmed by SAGE (x40) and by ISH for the four samples studied (Fig. 2bGo). The 21-hydroxylase (CYP21A2) mRNA is also overproduced in the APA according to SAGE (x2) and ISH for the seven samples studied (Fig. 2Go, b and c). The genes encoding enzymes, which catalyze the initial steps of steroidogenesis, were expressed at similar levels in the APA and ZG, according to SAGE (Fig. 2bGo).

Supply of cholesterol and electrons for steroidogenesis

Free cytosolic cholesterol is the substrate for steroidogenesis and may be obtained from three potential sources (Fig. 3aGo). We therefore analyzed differential expression of the genes involved in 1) capture of extracellular low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles, 2) endogenous biosynthesis, or 3) hydrolyze of intracellular esterified cholesterol stores (11). Interestingly, only the HDL receptor scavenger B1(SR-B1) gene was expressed more strongly in the APA than in the adjacent ZG according to SAGE (x5) and ISH for eight of the nine samples (Fig. 3Go, b and c). Expression of the other genes was not modified (Fig. 3bGo). These findings suggest that the cholesterol supply is increased through the HDL pathway in patients with PAL.




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  		FIG. 3. Differential expression of genes involved in the supply of cholesterol and electrons to the mitochondria. a, Schematic representation of the pathways of cholesterol and electron supply to the mitochondria. In black, genes expressed more strongly in APA than in adjacent ZG; in gray, genes with no differential expression. ADX, Adrenodoxin; CYB5, cytochrome b5; SRB1, scavenger receptor B1 (HDL receptor); LDLR, LDL receptor; POR, cytochrome P450 oxidoreductase. b, Differential expression of genes involved in supplying cholesterol and electrons to the mitochondria, as evaluated by SAGE and ISH, represented as described in Fig. 2Go. c, An APA (A and C) and its adjacent adrenal cortex (B) or ZG (D) from patient 4 after ISH using a scavenger receptor B1 (SR-B1) probe. A and B, Bright-field and adjacent dark-field photomicrographs showing strong labeling of the APA (A) and zona fasciculata (B), but not of the adjacent ZG (B). C and D, Higher magnification of the same APA and adjacent ZG, confirming differential expression. d, An APA (A and C) and the adjacent adrenal cortex (B) or ZG (D) from patient 7 after ISH using the POR probe. A and B, bright-field and adjacent dark-field photomicrographs show strong labeling of the APA (A) and zona fasciculata (B), but not of the adjacent ZG (B). C and D, Higher magnification of the same APA and adjacent ZG confirming differential expression. ZF, Zona fasciculata; C, capsule. Scale bar, 50 µm (A and B) and 10 µm (C and D).

Continued

 
Free cytosolic cholesterol was then transferred to the inner mitochondrial membrane by the specific transporter steroid acute regulatory protein (StAR) (18). The levels of mRNA for StAR and its partner, the peripheral benzodiazepine receptor, were similar in the two tissues (Fig. 3bGo).

The catalytic activity of cytochrome P450 enzymes requires electrons in addition to cholesterol, and three specific electron donor systems have been identified (Fig. 3aGo). We found differential expression of the genes for adrenodoxin according to SAGE (x2.4; Fig. 3bGo) and P450 oxidoreductase (POR) according to SAGE (x2) and ISH in six of eight samples (Fig. 3Go, b and d), but not for cytochrome b5. These results suggest that additional electrons are provided in APA via adrenodoxin and P450 oxidoreductase pathways.

Intracellular calcium tone is chronically stimulated in APA

Calcium is the principal intracellular messenger for aldosterone production in response to acute stimulation by angiotensin II or extracellular potassium variation (12). The increase in cytosolic calcium concentration induces rapid calcium capture by mitochondria, which is required for steroidogenesis, and the slower activation of calcium/calmodulin-dependent kinases, which have pleiotropic effects, including effects on gene transcription (Fig. 4AGo).



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  		FIG. 4. Differential expression of genes involved in calcium signaling. A, Schematic representation of the factors involved in the calcium signaling pathway identified in the SAGE libraries. In black, the genes overexpressed in PAL; in gray, genes with no differential expression. CALM2, Calmodulin 2; CALR, calreticulin; IP3, inositol 1,4,5-triphosphate; PLC, phospholipase C; SERCA3, sarcoplasmic reticulum Ca2+-adenosine triphosphatase 3. B, Differential expression of the genes involved in calcium signaling, as estimated by SAGE and ISH and represented as described in Fig. 2Go.

 
Interestingly, and for the first time in PAL, the SAGE transcriptome comparison of the two tissues indicates that several genes involved in calcium metabolism are more expressed in APA (Fig. 4BGo). According to SAGE, the calcium effector calmodulin 2 is more abundant in APA (x2); the endoplasmic reticulum calreticulin and calcium adenosine triphosphatase 3 (SERCA3) are also more abundant in APA (x3 and x9, respectively), suggesting that the calcium storage activity of the endoplasmic reticulum increases in patients with PAL. These changes may reflect an increased calcium signaling and intracellular calcium tone. However, ISH could not significantly confirm these results, reflecting both the inability of ISH to confirm tiny differences and the heterogeneity among APAs.

The possibility of an increased calcium signaling is supported by an additional observation: the mRNAs encoding the Gq protein-coupled receptors angiotensin II type 1 receptor (AT1R) and endothelin type B receptor (ETB), both of which activate calcium signaling, were more abundant in APA according to SAGE (Fig. 4BGo). ISH showed an important heterogeneity among APAs, but more than half overexpressed at least one of those two receptors.

Tumorigenic potential of APA

Although APA are benign tumors, we decided to investigate the expression of oncogenes and tumor suppressor genes. SAGE showed that several oncogene mRNAs were more abundant in the APA than in the adjacent ZG, including the mRNA for a Jun-binding protein JAB1 (x5), the avian myelocytomatosis viral oncogene v-MYC (x3), IGF-binding protein-2 (x4), teratocarcinoma-derived growth factor (TDGF1) (x6), and nephroblastoma overexpressed gene (NOV) (x1.5). However, ISH showed that the level of expression of v-MYC, IGF-binding protein-2, and NOV was highly heterogeneous (data not shown). It is not possible to draw firm conclusions concerning common primary mechanisms of tumorigenesis in APA from these data.

New clues to the genetic and functional pathogenesis of APA

Finally, three genes coding for proteins of unknown functions are overexpressed in APA. The mRNAs for hemopoietic stem/progenitor cells 176 (NM_016209), hypothetical protein MGC2198 (NM_138820), and multiple myeloma overexpression gene 2 (MYEOV2, AF487338), were more abundant in the APA according to SAGE (x6, x7, and x5, respectively; P < 0.05). ISH partially confirmed these differences (P = 0.09, 0.2, and 0.03, respectively; Fig. 5bGo). This overexpression may provide clues to the function of these genes and the pathogenesis of PAL.



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  		FIG. 5. Differential expression of poorly characterized genes. a, The differential expression of several poorly characterized genes, as estimated by SAGE and ISH and represented as described in Fig. 2Go. b, An APA (A and C) and the adjacent adrenal cortex (B) or ZG (D) from patient 2 after ISH using the fascin homolog 1 (FSCN1) probe. A and B, Bright-field and adjacent dark-field photomicrographs showing stronger labeling of the ZG (B) than of the APA (A) and zona fasciculata (B). C and D, Higher magnification of the same APA and adjacent ZG confirming differential expression. Scale bar, 50 µm (A and B) and 10 µm (C and D).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In this study we compared gene expression in APA and the adjacent ZG tissue. This strategy presents several advantages for studying aldosterone steroidogenesis in PAL. Firstly, the cell populations compared are both differentiated for aldosterone production. Most of the previous studies of gene expression in APA have used as reference the whole adrenal cortex, which mostly produces cortisol. This results in a nonappropriate comparison between cortisol and aldosterone steroidogenic tissues. Secondly, we compared a tissue with autonomous aldosterone production to a tissue in which aldosterone production is regulated by circulating angiotensin II. In the situation of adjacent ZG, circulating angiotensin II, and therefore aldosterone production, is low due to the negative feedback. Thus, the observed differences are independent of circulating angiotensin II levels and better reflect the steroidogenic alterations responsible for autonomous aldosterone production. Thirdly, the APA and ZG compared came from the same patient, eliminating interindividual variations.

Differences in gene expression were characterized by two complementary approaches: SAGE and ISH. SAGE is the most reliable and potentially exhaustive transcriptome analysis method for quantifying transcripts, even if the amount of starting material is extremely small, as for the ZG sample. However, this technique is limited by the sequencing capacity and does not allow the comparison of an extensive number of tissues. We used ISH to confirm the differences in gene expression observed by SAGE for several candidate genes, using several APA. Despite its limited value for quantification, we preferred ISH to RT-quantitative PCR, because this technique made it possible to clearly identify the different cell types of the adrenal cortex, such as ZG cells, which cannot be easily separated by microdissection in the absence of anatomical limits. In this study, the semiquantitative capacities of ISH were increased by 1) the normalization of hybridization conditions, because the two samples compared were present on the same slide; and 2) the stringent criteria used for the analysis (see Subjects and Methods). For many of the genes studied, ISH confirmed the SAGE data, increasing the reliability of our findings.

This transcriptome comparison of APA and adjacent ZG tissues provides an integrated view, without prior assumptions or experimental bias, of the molecular alterations in steroidogenic cells leading to the chronic, autonomous overproduction of aldosterone. By analyzing the expressions of various genes involved in steroidogenesis, cholesterol and electron supply to the mitochondria for steroidogenesis, and calcium signaling, we identified specific changes in each of these pathways.

Analysis of steroidogenic enzyme expression in APA found overexpression of aldosterone synthase mRNA, as previously reported (7, 8, 9, 19, 20), but also, and unexpectedly, of 21-hydroxylase. In a recent study, Bassett et al. (20) compared by microarray analysis and quantitative RT-PCR the expression profiles of the steroidogenic enzymes in APA and normal adrenal cortex. As in the present study, they demonstrated an increased expression of 21-hydroxylase in APA by RT-PCR, but not by microarray. No significant overexpression of 21-hydroxylase in APA was reported in previous studies (7, 21, 22). These discrepancies may be explained by the measurement of 21-hydroxylase in cortisol-producing cells, because the entire adrenal cortex was the reference tissue. For the two upstream steroidogenic enzymes, cholesterol side-chain cleavage and 3ß-hydroxysteroid dehydrogenase type II, we found no differential expression, in agreement with previous works (7, 22). Bassett et al. (20) report similar results by microarray, but found an overexpression of 3ß-hydroxysteroid dehydrogenase type II by RT-PCR. These data are concordant and suggest that overexpression of steroidogenic enzymes is limited to the two enzymes that produce aldosterone-specific steroids, i.e. steroids that cannot be 17-hydroxylated toward the cortisol pathway. Under these conditions, an explanation could be that the enzymes of the initial steps of steroidogenesis are not rate limiting for the aldosterone hyperproduction in PAL Another hypothesis suggested by several researchers (20, 23) is that steroid precursors may diffuse freely from the entire adrenal cortex to the adenoma and are converted to aldosterone in APA. This hypothesis has little experimental support.

Because this study concentrates on aldosterone-producing tissues, our data do not generate any information about differences in steroidogenic enzymes with other adrenocortical adenoma, and therefore, we cannot speculate about the specificity of the APA steroidogenesis expression profile. Other previous publications suggest specific steroidogenic profiles according to the hormonal secretion (7, 19, 20); on the one hand, overexpression of aldosterone synthase is certainly the most preeminent phenotypic trait of APA; on the other hand, 17{alpha}-hydroxylase/17–20 lyase and 11ß-hydroxylase are the two specific enzymes for cortisol production. However, in APA, the distinction between the aldosterone and cortisol steroidogenic pathways is not so clear: hybrid steroids such as 18-hydroxycortisol are found in patients with PAL (24), and 17{alpha}-hydroxylase/17–20 lyase and 11ß-hydroxylase are expressed at various levels in APA (7, 19, 20). Accordingly, our SAGE data indicate that both 17{alpha}-hydroxylase/17–20 lyase and 11ß-hydroxylase are expressed in APA. By ISH, we found that 11ß-hydroxylase was overexpressed in a majority of APA compared to adjacent ZG, but not all. Together, these data suggest that APA may also produce cortisol.

To synthesize steroids, adrenal cells can either synthesize or capture cholesterol through specific receptors of the lipoprotein particles (11). Our results provide the first evidence for a preferential capture via the HDL pathway in APA cells, rather than the LDL pathway or endogenous synthesis (Fig. 3aGo).

Another limiting step of the acute steroidogenic response in vitro is cholesterol transport through the inner membrane of mitochondria. This step involves the protein StAR, which is a key regulator (18). Therefore, it is surprising to observe only a slight, but nonsignificant, overexpression of StAR in APA (P = 0.26). Similar observations were made in previous works comparing APA and the entire normal adrenal cortex (20, 25, 26). These results confirm that the increase in StAR activity is not obtained by an increase in its transcription, but by posttranscriptional mechanisms, including protein synthesis and translocation to the inner mitochondrial membrane, as shown in vitro (27, 28).

Very little is known about electron supply to the cytochrome P450 steroidogenic enzymes in PAL. In acute stimulation of steroidogenesis, electron providing is a rate-limiting factor (29). In this study, overexpression of adrenodoxin and P450 oxidoreductase in APA suggest the involvement in PAL of two of the three known pathways (30). Inactivating mutations of P450 oxidoreductase have been recently shown to be responsible for complex congenital syndromes associated with steroidogenic deficiency (31), but we provide here the first demonstration of P450 oxidoreductase involvement in PAL.

Calcium is the principal intracellular mediator of the acute stimulation of aldosterone production by angiotensin II and potassium. Interestingly we observed in APA a coordinated overexpression of several mRNAs encoding calcium-activating membrane receptors, calcium-signaling effectors, and proteins involved in endoplasmic reticulum calcium storage. This observation opens a new field of investigations to determine whether a chronic increase in aldosterone production in PAL may result from an increase in calcium tone.

We reproducibly found calmodulin 2 overexpression in APA. Calmodulin has been shown to play a key role in regulating aldosterone production after acute stimulation in in vitro models. For example, the direct injection of calmodulin into adrenal cells increases steroidogenesis (32), and calmodulin antagonists block the stimulation of aldosterone production induced by angiotensin II, ACTH, and K+ (33, 34, 35).

Activation of the calcium-signaling pathway in PAL may be explained by an abnormal activity of Gq protein-coupled membrane receptors. Interestingly, our data show unambiguously and for the first time an overexpression of AT1R and/or ETB in half of the APA compared with adjacent glomerulosa. Previous works have found an overexpression of AT1R (36, 37, 38) and a repression of ETB (39, 40, 41) in APA compared with the entire adrenal cortex, but these results reflect more the expression in the reference tissue (42). Activation of AT1R or ETB may result from an overproduction of these Gq-coupled receptors, as observed in this study, but also from an increase in their basal activity and/or from the local increase in their receptor ligand.

This chronic calcium activation may be the initial event responsible for the other major transcriptome alterations described above. According to previous in vitro studies, 1) among the steroidogenic enzymes, the calcium-calmodulin pathway activates both aldosterone synthase (43, 44) and 21-hydroxlyase expression (45), whereas calcium has little or no effect on upstream enzymes (28, 45); 2) acute calcium activation by angiotensin II results in an increase in HDL receptor with no increase in LDL receptor (46); and 3) intracellular calcium does not modify Star mRNA expression (27, 28). Therefore these in vitro data draw an expression pattern, which exactly matches that observed here.

In summary, the activity of the calcium-signaling pathway seems to increase in APA and fits nicely with the positive regulatory effects of angiotensin II and potassium on aldosterone production. In contrast, we found no mRNAs coding for proteins involved in cAMP signaling overproduced in APA. This observation is apparently difficult to reconcilliate with the exquisite sensitivity of APA to ACTH for aldosterone production (47, 48). The sensitivity of APA to ACTH is associated to the overexpression of ACTH receptor (MC2R) in APA (48), which is able to induce a calcium signal in glomerulosa cells (49). The absence of cAMP-signaling effectors in APA should be interpreted with caution, because many of the corresponding mRNAs may be produced in amounts too small to be detected by our SAGE screening. This limit of detection by SAGE in our conditions also concerns the adrenal cortex-specific transcription factors that have been previously described (10).

In conclusion, our data strongly suggest the coordinated overexpression of genes involved in steroidogenesis and in the supply of cholesterol and electrons. These modifications may be due to a chronic, permanent increase in calcium tone, the mechanism of which should be investigated. They illustrate how integrated sequence information from human pathological tissue contributes to the understanding of physiopathology and may therefore provide an original starting point for future mechanistic studies.


   Footnotes
 
This work was supported by grants from Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, University René Descartes, Association pour la Recherche sur le Cancer, Ligue contre le Cancer (Comité de Paris and Ligue Nationale), and Fondation de France (to European Community). This study was supported in part by Programme Hospitalier de Recherche Clinique (PHRC) Grant AOM02068 and grants from Institut National de la Santé et de la Recherche Médicale and Ministère Délégué à la Recherche et des Nouvelles Technologies for the COMETE Network.

First Published Online October 4, 2005

Abbreviations: APA, Aldosterone-producing adenoma; AT1R, angiotensin II type 1 receptor; ETB, endothelin type B receptor; HDL, high-density lipoprotein; ISH, in situ hybridization; LDL, low-density lipoprotein; PAL, primary aldosteronism; POR, P450 oxidoreductase; SAGE, serial analysis of gene expression; StAR, steroid acute regulatory protein; ZG, zona glomerulosa.

Received June 15, 2005.

Accepted September 28, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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