A Review of Markers of Zona Glomerulosa and Aldosterone-Producing Adenoma Cells

Primary aldosteronism (PA) is the most common form of secondary hypertension comprising more than 11% of the patients referred to specialized centers for hypertension.¹ The term ‘primary’ denotes our ignorance of the underlying cause(s)/mechanism(s),² due largely to the lack of ideal models for in vitro and ex vivo investigation of the regulation of aldosterone production. In fact, we still rely on the H295R line and its derived clone HAC15; as adrenocortical carcinoma cells, they may not closely recapitulate the physiological and pathophysiological regulation of aldosterone secretion. Until recently the lack of biomarkers specific for aldosterone-producing adenoma (APA) cells for identification and isolation and, thereby, ex vivo investigation, has been a major hurdle in this field. We herein review available knowledge on zona glomerulosa (ZG) and aldosterone-producing cell markers with the aim of providing updated information on the state-of-the art on this topic. Table 1 summarizes the information that could be gathered for each candidate marker regarding its specificity, intracellular localization, usefulness and drawbacks for isolating aldosterone-producing cells from the ZG and APAs. For the sake of clarity the text below follows the order of markers shown in this Table. For further roles and functions of the markers see Supplemental data and Figures.

Table 1.

Markers of aldosterone-producing cells: specificity, localization, usefulness, and drawbacks.

Marker	ZG specificity	ZF specificity	APA	Cellular localization	Useful for isolation of A-P cells	Drawbacks
CYP11B2	+++	−−	+++	Mitochondria	N	Inconsistently over-expressed in APAs
TASK-2 channels	+++	+−	++	Cell membrane/Cytoplasm	N	Underexpressed in APA
KCNJ5 channels	+++	+−	=	Cell membrane	N	Expressed also in tissue adjacent to APA
CD56	+++	−−	+++	Cell membrane	Y	Faintly expressed in ZF cells
Dab2	+++	−−	+++	Cytoplasm	N	Not expressed in all ZG cells
Wnt/β-catenin pathway	+++	−−	+++	Cell membrane/Cytoplasm/Nucleus	N	Also expressed in adjacent tissue to APA
Sonic Hedgehog Signaling (SHH)	+++	−−	+++	Cell membrane	N	Also expressed in adjacent tissue to APA
AT1	+++	+−	+++	Cell membrane	N	Also expressed in adjacent APA tissue
DACH1	+++	−−	−−	Cytoplasm/Nucleus	N	Not overexpressed in APAs
LGR5	+++	−−	−−	Cell membrane	N	Not overexpressed in APAs
VPREB3	+++	+−	+++	Cytoplasm/Nucleus	N	Also expressed in adjacent tissue to APA
NPNT	+++	−−	+++	Extracellular localization	N	Only expressed in ZG-like APA
NEFM	+++	−−−	+++	Cytoplasm	N	Only expressed in ZG-like APA

Legend: AP: aldosterone-producing, Y= yes; N= No; ZG = zona glomerulosa; ZF= zona fasciculata. For abbreviations of marker names see text.

Aldosterone synthase (CYP11B2) as a biomarker of the aldosterone-producing cells

The adrenocortical zona glomerulosa (ZG) is characterized by cells lying directly beneath the capsule and arranged in clusters or arches (‘glomus’ in Latin means ‘ball’), and is the main site of aldosterone synthesis. Rodent ZG comprises 4-to-6 layers of cells in a continuous band occupying 2–15% of the gland volume depending on salt intake. In humans its size varies with age: in infants and young adults the ZG resembles the rodent adrenal with a relatively homogeneous layer of ZG cells, while after the fourth decade it appears as a discontinuous layer comprising no more than 5% of the total adrenocortical volume.³

The recent development of specific antibodies against human 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) now allows an accurate localization of the cells producing cortisol and aldosterone in the normal adrenal cortex and APA.³ These antibodies show that some human APAs contain not only CYP11B2 expressing cells, but also CYP11B1-positive cells.^4–7 In some APAs CYP11B2 or CYP11B1 are expressed in different cells; in others cells express both CYP11B1 and CYP11B2.^4–7 These findings challenge the classic view of APA as a single, well demarcated nodule comprising only cells producing excess aldosterone.⁴ Clusters of CYP11B2 positive cells, called aldosterone-producing cell clusters, APCCs, have been identified in the subcapsular area of adrenals with and without an APA,⁸ including some without high blood pressure.⁹ This suggests no clear boundary between the APA and adjacent ZG tissue, and that subcapsular micronodules might be the initial foci for the development of APA; this contention remains to be proven.⁴

Our initial observation that compared to the normal adrenal tissues, CYP11B2 gene expression was not increased in some APAs fulfilling the 4 corners’ criteria,¹⁰ confirmed by Zennaro et al.,¹¹ along with the aforementioned identification of CYP11B1-positive cells within the APA, challenged the hypothesis of CYP11B2 as a unique marker of APA.^4,6,7,12 Thus, APA entirely of cells expressing abnormally high levels of CYP11B2 are at one end of the spectrum and APA with CYP11B2 expression similar to the normal ZG cells at the opposite end. In patients with AVS-proven unilateral production of aldosterone but an adenoma that does not clearly express CYP11B2, hyperaldosteronism may be sustained by unilateral expansion of aldosterone-producing cell number and/or by concomitant cortical APCC.^9,13,14

K⁺ channels

TASK-1 and TASK-2 are highly expressed in the normal human adrenal cortex; in mice deletion of TASK-1 and TASK-3, or both, results in hyperaldosteronism. Normally TASK-1/TASK-3 hyperpolarize the cells and blunt aldosterone production (see Supplemental data).¹⁵ TASK-2 is under-expressed in APAs compared with normal adrenal cortex; moreover, lowered TASK-2 activity markedly increases CYP11B2 gene expression and aldosterone production in H295R cells,¹⁶ thus making this channel a candidate marker of APAs. However, its down-regulation in APA precludes use of TASK-2 as a tool for APA cell isolation: its possible use is as a ‘negative selector’, e.g. to remove non-APA cells from a specimen.

Several mutations in other genes regulating ion homeostasis and cell membrane potential have been found in sporadic APAs and familial forms of PA (reviewed in¹⁷). These mutations do not involve all APAs; for example the most common are the Kir3.4 channel (KCNJ5) mutations that occur in 30% to 70% of the APA with wide geographic variations;^18–21 ion channel mutations, therefore, cannot be regarded as universal markers of these tumors. At variance with an early suggestion, HAC15 human adrenal cortical cells with mutations involving the Kir3.4 channels do not increase proliferation, but rather the opposite.^22,23 Finally, different mutations can be harbored within the same APA.^21,24 Thus, the somatic mutations of K channels cannot be regarded as markers of APA, since by definition a marker should be a trait of the disease. However, recent data suggest that some Kir3.4 mutations could show sensitivity to certain drugs, which may thus be an indication of the presence of mutated APA.²⁵

CD56: the intercellular glue

We found CD56 immunostaining of the neural cell adhesion molecule (NCAM), known as CD56 (NM_000615), which fades moving toward the ZF, is strong in the ZG and APAs. We thus suggested that CD56 may be a marker of ZG and APA (see Supplemental data).²⁶ We also showed that CD56, located on the cell surface, can be used to isolate aldosterone-producing cells from ZG and APAs on immunobinding,²⁶ thus distinguishing CD56-positive and CD56-negative cells. CYP11B2 gene expression levels and aldosterone concentrations in the culture media are markedly higher in the former than in the latter, evidence that CD56 is a marker of ZG and aldosterone-producing cells.²⁷ Thus, CD56 is a membrane marker of ZG and APA cells and a useful tool to isolate these cells, with the caveats that it is not expressed in all ZG cells and is highly expressed in the adrenal medulla.²⁷

Dab2: the driver of directional motor in the cell

Dab2 (NM_001343) is a 96kD phosphoprotein isolated from murine macrophages involved in colony stimulating factor (CSF)-1 signaling and mitogenic pathways (see Supplemental data).²⁸ In the rat it is expressed in the adrenal ZG, but not the ZF-ZR or medulla.²⁹ In H295R cells Dab2 overexpression yielded two alternatively spliced Dab2 isoforms (of 100 KDa and 75), which enhanced Angiotensin II-stimulated aldosterone secretion.²⁹ Low-salt diet treatment doubled Dab2 immunoreactivity of the 100 KDa Dab2 isoform, along with enhanced ZG proliferation, suggesting that the longer isoform acts as a sensor of [Na⁺] changes.²⁹ In the adrenal cortex Dab2 co-localizes with CYP11B2,^29,30 whereas only the outer layer of APCC expresses Dab2.³⁰ Like CD56, Dab2 is expressed only in differentiated ZG cells: its expression is lost when ZG cells trans-differentiate into ZF cells, and its intracellular location does not allow immunobinding separation. Dab2 is expressed in ZF-like APA cells,³⁰ but about a third of such APAs are negative.³¹ Although the expression of Dab2 in differentiated ZG, but not in mature ZF cells, suggests it could be a marker for ZG and APA cells, the lack of Dab2 in a third of APAs questions this contention. Elucidation of Dab2 function (Figure 1, panel A) could illuminate its pathophysiologic role and heterogeneous expression in APAs.

Figure 1. Activation of Dab2-, Wnt/β-catenin-, SHH- and Angiotensin II (Ang II)-mediated pathways in aldosterone secretion.

A. An increased expression of Dab2 was found in zona glomerulosa (ZG) and APA; whether it is due to reduced destruction of the Dab2/clathrin complex is unknown. Possible pathways by which Dab2 regulates CYP11B2 expression are indicated with dotted lines: a) direct triggering of CYP11B2 transcription; b) activation of TGFβ1 receptor/Smad-mediated pathways; c) activation of Wnt/β catenin pathway. B and C. The canonical (left) and non-canonical Wnt signaling pathways (right) are triggered by the binding of Wnt to the Frizzled serpentine receptor Fzd. In the canonical pathway the complex Wnt/Fzd binds the co-receptor LRP5/6 and, after recruiting the dishevelled protein Dsh, induces disassembling of the β-catenin complex formed by axin, Gsk3, APC and Ck1. This causes translocation of β-catenin to the nucleus, where it binds to the TCF/LEF1 proteins and triggers transcription of genes involved in growth and cell fate. In the APA this pathway favours CYP11B2 expression, directly via the nuclear receptors NURR1 and NURR7, or indirectly by enhancing the expression of AT1 receptors and the conversion of progesterone into 11β deoxycorticosterone via CYP21. In the non-canonical, Wnt, after binding to Fzd and recruiting Dsh, activates Rho associated kinase pathway and/or phosphokinase C (PKC), thereby regulating cytoskeleton and Ca²⁺ release from intracellular stores. Involvement of the non-canonical Wnt was suggested, but not demonstrated, in APA. D. The SHH glycoprotein binds the 12-transmembrane protein patched 1 (PTCH1) that, in turn, inhibits the smoothened receptor (SMO) leading to activation of GLI, and downstream of genes involved in cell growth and differentiation. SHH is barely detectable in the normal ZG, but markedly, albeit heterogeneously, expressed in APAs. E. Ang II, after binding to AT1R receptor, activates a variety of intracellular signalling pathways, including phospholipase C (PLC) via G protein subunit q/11 (Gq/11), protein kinase C (PKC), calcium/calmodulin-dependent kinases (CaMK), mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase 1 and 2 (ERK1/2), Janus kinase (JAK)/signal transduction and activators of transcription (STAT), which lead to phosphorylation of transcription factors (TF) and transcription of genes involved in steroidogenesis and cell growth.

Wnt/β-catenin pathway

Wnt (Wingless-related integration site) are secreted proteins that control growth and stem cell renewal, acting via canonical and non-canonical Wnt pathways (see Supplemental data; Figure 1, panels B and C). The finding of immunostaining for β-catenin, the central signaling molecule of the canonical Wnt pathway, at the nuclear level in a small series of APAs with high expression of CYP11B2 suggested that Wnt/β-catenin may regulate aldosterone production.³² Aldosterone is increased in transgenic mice with constitutive activation of β-catenin in the adrenal cortex.³³ Silencing of β-catenin in H295R cells decreased basal aldosterone production and blunted the aldosterone response to Angiotensin II.³² Activating catenin beta 1 gene (CTNNB1) mutations causing Wnt/β-catenin pathway activation are associated with the adrenal malignancy³⁴ and two series of APA.^35,36 β-catenin enhanced aldosterone production in these APAs at several points: it increased expression of the Angiotensin II type 1 receptor (AT1-R), conversion of progesterone into deoxycorticosterone (the substrate for CYP11B2) and expression of the nuclear receptors NURR1 and NURR7.³²

Wnt/β-catenin pathway activation was associated with down-regulation of secreted frizzled-related protein 2 (SFRP2) in APAs.³² The SFRP2 knockout mouse shows increased plasma aldosterone levels, ectopic Dab2 plus β-catenin positive cells in the cortex and the central region.³² Collectively these data indicate that the Wnt inhibitor SFRP2 causes dysregulation of the Wnt/β-catenin pathway, and suggests that SFRP2 and Wnt/β-catenin could be markers of APA. The finding, however, of constitutive Wnt/β-catenin signaling in the entire peritumoral adrenal cortex,¹¹ in micro- and macronodular hyperplasia and in non-aldosterone-producing adrenocortical tumors challenges this contention.^32,35 Thus, whether SFRP2 and Wnt/β-catenin can isolate aldosterone-producing cells and/or identify APA is doubtful.

Sonic Hedgehog Signaling (SHH)

The SHH gene (NM_000193) codes for a glycoprotein involved in cell growth and differentiation (see Supplemental data; Figure 1, panel D).^37,38 SHH components are expressed in the entire adrenal cortex in children and only in the outer subcapsular ZG in adults,^11,39,40 suggesting that SHH may be a marker of stem/precursor cells in the adrenal cortex. SHH is barely detectable in the outer normal ZG, but is expressed in APAs with homogeneous or heterogeneous patterns;¹¹ SHH is also expressed in peritumoral tissue and in the hyperplasic ZG with a pattern of expression similar to CYP11B2 and Dab2.¹¹ This abnormal expression was associated with activation of a cluster of genes (HMGA1, TUBB3, GATA2, ZNF9, UNG, CCND2, STAT1) involved in SHH pathways.¹¹ As APA and adjacent ZG present features of stem/precursor cells, the re-expression of these fetal genes possibly represents excessive ZG cell proliferation and APA formation.¹¹ SHH is, however, unlikely to be a good marker of aldosterone-producing and APA cells, given its expression in both APAs and peritumoral tissue.

Angiotensin II type 1 (AT1) receptor

Receptors for Angiotensin II, a well-established stimulus of aldosterone secretion (Figure 1, panel E) and cell growth,^41,42 were reported two decades ago.^43,44 Only recently, however, a systematic investigation of human adrenal specimens has shown that type 1 receptors (AT1) are by far the most predominantly receptor in both normal adrenal cortex and APA; AT2 are 10-fold less expressed in the normal adrenal cortex, and even less in APAs.⁴⁵ AT1 is also found in ZF and the tissue adjacent to APAs, indicating that AT1 is not exclusively involved in regulation of aldosterone. These observations together with the low expression of AT2 and the receptor for alamandine, a derivative of Ang (1–7), in both normal ZG and APA invalidate these receptors as markers.

DACH1: a CYP11B2 suppressor

The nuclear protein Dachshund family transcription 1 (DACH1, NM_004392.6) acts as a tumor suppressor by binding to Smad4.^46,47 DACH1 was found to be 10-fold more expressed in ZG than in ZF and APA cells;⁴⁸ its silencing in H295R cells increases aldosterone synthesis and over-expression decreases it. Stimulation of primary human adrenal cells with Angiotensin II to markedly increase CYP11B2, leading to 2.4-fold increase in aldosterone production, blunted DACH1 gene expression.⁴⁸ Hence, DACH1 regulates ZG cell function⁴⁸ through mechanisms yet unknown. Since overexpression of DACH1 increased SMAD4 expression and enhanced the TGF-β1 signaling pathway, it was suggested that activation of DACH1/TGF-β1 pathway switches off aldosterone production, and that such an inhibitory pathway is blunted in APA (Figure 2).⁴⁸ Overexpression of TGFβ1 in mice decreases aldosterone production, and decreased expression of TGFβ1 increases aldosterone production.⁴⁹ Whether DACH1 can be a negative marker of ZG and APA cells remains to be proven.

Figure 2. Regulation of aldosterone secretion by DACH1 in normal ZG and APA cells.

DACH1 was found 10-fold more expressed in ZG than in ZF and APA (panel A). Angiotensin II blunts expression of DACH1 and thus increases CYP11B2 transcription and aldosterone production (panel B); contrarily, overexpression of DACH1, by increasing SMAD4 expression and activating the TGF-β1 signalling, turns off aldosterone production in the normal ZG. Blunted expression of DACH1 (likely independently from Angiotensin II) could activate aldosterone production in APA (panel C).

LGR5 suppresses CYP11B2 in APA

LGR5 (NM_001277227.1) is a 907-aminoacid protein involved in growth and proliferation (see Supplemental data). A transcriptome analysis of laser capture microdissected ZG and ZF cells from adrenal cortex adjacent to APA or pheochromocytoma showed that expression of LGR5 was 25-fold greater in ZG than in ZF and was the ‘top hit’ among 213 over-expressed genes in ZG compared to ZF.⁵⁰ A radial streaking of LGR5 into ZF was observed in some serial sections, presumably reflecting the centripetal migration of ZG cells during development.⁵⁰ Expression of the gene coding for the LGR5 cognate ligand R-spondin-3 (RSPO3) was also up-regulated 5-fold in ZG compared to ZF. In addition, 18 of the 213 genes more expressed in the ZG than ZF encoded proteins associated with the Wnt signaling pathway (see later); translation of some of these mRNA into effector proteins was confirmed with immunohistochemistry.⁵⁰ LGR5 showed specific ZG expression and co-localization with β-catenin, LEF1, the nuclear transcription factor activated by β-catenin, c-Jun, the target of the polarity non-canonical Wnt pathways, and phospho-c-Jun.⁵⁰ Its silencing in H295R cells and normal primary adrenal cells increases both CYP11B2 expression and aldosterone production; it also increases activity of the Wnt transcription factors TCF/LEF and AP1/Jun, indicating that LGR5 blunts aldosterone synthesis via Wnt pathways (Figure 3). Conversely, over-expression of LGR5 agonist cognate RSPO3 has opposite effects.⁵⁰ The lower expression of LGR5 observed in APA cells than in the adjacent ZG also supports the contention that LGR5 blunts aldosterone synthesis, by suppressing Wnt pathway, whose activation results in aldosterone synthesis (see later).^32,33,50 Whether LGR5 can be used as a tool to isolate APA from surrounding non-APA cells remains to be investigated. Whether the LGR5 pathway is a feasible therapeutic target for mitigating hyperaldosteronism is an intriguing possibility.

Figure 3. Regulation of aldosterone secretion by LGR5 in normal ZG and APA cells.

LGR5, by acting as a receptor for R-spondins (RSPO), amplifies the effect of Wnt/β-catenin signaling. After binding of the complex Wnt/Fzd to LRP5 and recruitment of Dsh, the β-catenin complex disassembles, thus allowing translocation of β-catenin to the nucleus, where it regulates gene transcription (see Figure 1 for details on Wnt/β-catenin signalling). It was posited that high levels of LGR5 in the ZG prevent abnormal aldosterone synthesis, whereas lower levels in APA would be permissive.

VPREB3

Pre-B lymphocyte protein 3 (VPREB3) is expressed in APAs and adjacent tissues, but not in cortisol-producing adenomas (see Supplemental data).⁵¹ In the normal cortex it occurs mainly in the normal ZG, with only few positive cells in ZF.⁵² The co-localization of VPREB3 with CYP11B2 and the increased expression VPREB3 in H295R cells after Angiotensin II stimulation suggests that VPREB3 could be a marker of aldosterone-producing cells.⁵² Nevertheless, the expression of VPREB3 in some ZF cells and the correlation between expression of VPREB3 and HSD3B2, but not HSD3B1, does not support VPREB3 as marker of ZG and APA cells.

Visinin like 1 (VSNL1)

VSNL1 (NM_003385.4) is a protein modulating intracellular Ca²⁺ signaling pathways (see Supplemental data). Overexpression of VSNL1 upregulates basal and Angiotensin II-stimulated CYP11B2 expression in the H295R cells.⁵³ VSN1 was reported to be highly expressed in the rat adrenal ZG, to be upregulated in APAs,⁵³ and to be acutely downregulated by ACTH.⁵⁴ APAs harboring KCNJ5 mutations overexpress VSNL1 compared to wild-type APAs, resulting in inhibition of Ca²⁺ induced apoptosis. The role(s) of VSNL1 in adrenal ZG physiology, and whether it is a marker of aldosterone-producing and APA cells, remains to be fully explored.

Nephronectin (NPNT)

Nephronectin (NM_001184690.1) is an extracellular matrix protein that has been recently described in the normal adrenal cortex, with higher expression in ZG than in ZF cells (see Supplemental data).⁵⁵ The expression in ZG-like APAs, but not in the adjacent tissue, suggests that NPNT could play a role in APA, a contention supported by the increased, or reduced, CYP11B2 expression levels after overexpressing or silencing NPNT gene, respectively, in H295R cells. Modulation of Wnt caused parallel changes of NPNT, and vice versa, suggesting a bidirectional regulation between Wnt and NPNT. NPNT silencing caused apoptosis through reduction of BCL2, a prosurvival factor, suggesting a role of NPNT in the adrenal growth. Why NPNT is over-expressed only in ZG-like, but not ZF-like APAs, is totally unknown.

Neurofilament medium polypeptide (NEFM)

The NEFM gene (NM_005382), which encodes the medium neurofilament protein, was recently found in normal ZG and ZG-like APAs, but apparently not in normal ZF or in ZF-like APAs (see Supplemental data).⁵⁶ Silencing of NEFM in H295R cells increased aldosterone production and decreased CYP11B2 and NR4A2 mRNA expression levels, and also enhanced cell proliferation, suggesting that NEFM suppresses aldosterone synthesis and growth.⁵⁶ NEFM interacts with dopamine receptor D1, which stimulates aldosterone secretion in both ZG and ZG-like APAs cells.⁵⁶ NEFM, by binding D1 receptor and internalizing it, promotes its desensitization and therefore blunts aldosterone secretion.⁵⁶ Because of the specific expression in ZG-like APAs, NEFM could be useful for identification of ZG-like APAs.

3β-hydroxysteroid dehydrogenase/isomerase (3βHSD)

The two isoforms HSD3B1 and HSD3B2 share 93.5% identity at the amino acid sequence (see Supplemental data).⁵⁷ Immunohistochemistry with specific monoclonal antibodies showed that HSD3B1 is expressed in ZG, but not in ZF, whereas HSD3B2 is equally expressed in ZG and ZF, and also in APA. In APA, HSD3B1 is expressed at low levels, but correlates with the expression of CYP11B2.⁵⁸ Hence, the isoform HSD3B1 could be a promising marker for APA, a hypothesis that deserves further specific investigation.

Nuclear factor erythroid-derived 2 (NRF2) and reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH)

Analysis of genes differentially regulated in ZG versus ZF showed overexpression in ZG versus ZF, and also in APA versus ZG, of NRF2 (NM_006164.4), a transcription factor that regulates expression of anti-oxidative proteins.⁵⁹ An involvement of this pathway is consistent with the increased levels of NOx2 and p22phox, which forms the catalytic core of NADPH, in APA patients.^60,61 While probably relevant for the pathophysiology of APA, the lack of data currently precludes use of either NRF2, or NADPH as markers of aldosterone-producing cells.

Conclusions and perspectives

For decades expression of CYP11B2 was thought to be the signature of ZG and APA cells, but this notion turned out to be fallacious. Hence, the search for specific markers of ZG and APA cells was initiated. By using advanced techniques different laboratories have made a tremendous effort to identify genes and proteins over- or under-expressed in ZG and APA cells that could be involved in steroidogenesis or cell growth. The results obtained thus far, although promising, raised several questions that still await answers. However, waiting for ‘the marker’ of aldosterone producing cells, a set of markers that can be reasonably used to diagnose an APA include CD56 and Dab2. This is because both are markedly expressed in ZG and APA, with the former also expressed in a percentage of ZF cells and the latter missing in some ZG cells. Combination of the two markers would allow identification of all aldosterone producing cells. Moreover, since CD56 is expressed at the cell membrane level, it can be successfully used to isolate aldosterone producing cells for research purposes.