Considering their enzymatic properties and expression levels in murine adrenal cortex, Akr1b7 and Akr1b8 are considered as the main isocaproaldehyde reductase and 4-HNE reductase, respectively, while Akr1b3 could rather participate in the elimination of these toxic compounds in basal physiological conditions (27–29). Moreover, Akr1b3 also displays 9-,11-endoperoxide reductase activity that, when coupled to COX-1 (cyclooxygenase type 1), allows prostaglandin F2α (PGF2α) synthesis in adrenal cortex and medulla (see below).

Despite all these evidences upon Akr1b3 involvement in both lipid aldehyde detoxification and PGF2α synthesis, *in vivo Akr1b3* gene invalidation (*Akr1b3*−*/*− mice) did not highlight any phenotype related to adrenal gland (30, 31). The lack of adrenal dysfunction in *Akr1b3*−*/*− mice may result from the redundancy of enzymatic properties carried by the other murine isoforms expressed in the gland (Akr1b7 and Akr1b8) that could then compensate Akr1b3 loss.

TABLE 3 | Localization and regulation of AKR1B in adrenal gland.

*a Intra-adrenal tissue localization was not specified.*

*n.d., not determined; NB, Northern Blot; WB, Western Blot; RT–PCR, reverse transcription–polymerase chain reaction; ISH, in situ hybridization; IHC,*

*immunohistochemistry; n.d., not determined.*

In human adrenal gland, *AKR1B1* transcripts have been initially detected using RNA master blot (1) (**Table 3**). Thereafter, using immunohistochemistry, we confirmed those results and demonstrated that AKR1B1 expression pattern is restricted to the cortex of adrenal gland (27). Treatment of the human adrenocortical tumor cells NC1-H295 with forskolin (adenylyl cyclase inducer) allowed us to suggest that similar to the murine isoform Akr1b7, *AKR1B1* expression was sensitive to ACTH (32) (**Table 3**). The molecular mechanisms and *cis*-acting elements responsible for ACTH/cAMP responsiveness of *AKR1B1* gene have not been investigated to date (**Figure 2**). Analysis of *AKR1B1* expression in stress-related disorders was not explored to date. Moreover, analysis of adrenal samples from Cushing's disease (ACTH-producing pituitary tumor) revealed unchanged mRNA levels of *AKR1B1* gene (32).

Based on its enzymatic properties, AKR1B1 has long been considered as the sole isocaproaldehyde reductase in the human adrenal gland (33) (**Figure 3A**; **Table 4**). Interestingly, unlike murine Akr1b7 isoform, NADPH-dependent isocaproaldehyde reductase activity carried by AKR1B1 was inhibited by tolrestat, a potent and specific aldose reductase inhibitor belonging to the carboxylic acids group of AR inhibitors (13, 29, 33). We demonstrated that AKR1B1 was also able to convert PGH2 into PGF2α (34) (**Figure 3C**; **Table 4**). This 9-,11-endoperoxide reductase activity is also strictly NADPH-dependent and inhibited by tolrestat.

We observed that in the human adrenal gland, AKR1B1 and the cAMP-inducible COX-2 isoform were co-localized in steroidogenic cortical cells (27) (**Table 3**). Then, we can consider that human adrenal cortex has the potential to produce PGF2α in response to ACTH surge. The shared properties of human AKR1B1 and mouse Akr1b7, such as hormonal regulation and reductase activity toward common substrates, prompted us to postulate that they can be considered as functional orthologs

protein 1 binding site; ARE, antioxidant response element; C/EBP, CCAAT enhancer binding protein binding site; Sp1, selective promoter factor 1 binding site.

at least in the adrenal cortex (32). Moreover, increased levels of *AKR1B1* transcripts in human were observed in adrenocortical adenomas harboring glucocorticoid autonomous hypersecretion (32). The possibility that AKR1B1 prostaglandin F synthase (PGFS) activity could participate in an intra-adrenal feedback loop between endocrine activities of cortex and medulla in human adrenal gland remains to be explored.

Given the high expression of *AKR1B1* in the adrenal cortex, we evaluated alterations in its expression in association with human adrenal disorders. The relative abundance of *AKR1B1* mRNA was decreased in adrenocortical carcinomas (ACC) when compared to benign tumors, Cushing's hyperplasia, or normal adrenals (32). These data were reinforced by de Reyniès et al., who demonstrated that decreased *AKR1B1* expression was associated with malignancy using an unsupervised clustering analysis of the human adrenal tumors transcriptome (35). This identified AKR1B1 as a potential negative marker for adrenocortical malignancy.

### Akr1b8/AKR1B10: Phylogeny, Regulation, and Enzymatic Specificities

*Akr1b8* mRNA was initially detected in both fetal and adult murine adrenal cortex but remained undetected in the medulla by *in situ* hybridization (2). In fibroblasts as well as in adrenocortical Y1 cell line, *Akr1b8* was previously shown to be controlled by the FGF and phorbol myristate acetate (PMA) (23, 28). On the contrary, dexamethasone-induced ACTH suppression did not influence *Akr1b8* mRNA and protein accumulation (27), suggesting that its biological function did not seem to be related to the ACTHdependent steroidogenic activity present in adrenocortical cells (**Table 3**). Interestingly, in digestive tract organs, such as liver and small intestine, *Akr1b8* gene was recently showed to be a target of NF-E2-related factor2 (Nrf2), which mediates transcriptional response to oxidative stress by binding to antioxidant response element (ARE) sites (36). As expected, *Akr1b8* expression was downregulated in digestive tract from Nrf2 knockout mice.

adrenal function remains unknown so far. AKR1B isoforms endowed with each of these activities are indicated in green.

*n.d., not determined.*

*Reference: a (33); b(40); c (28); d(11); e (12); f (34); g (14).*

However, no adrenal phenotype was described in these mice, and neither oxidative stress nor Nrf2 have been involved in adrenal *Akr1b8* expression so far.

Previous studies demonstrated that some AKR1B proteins showed efficient catalytic activity for the reduction of the abundant and highly reactive lipid-derived aldehyde 4-hydroxy-2-nonenal Pastel et al. AKR1B in Adrenal

and the phospholipid aldehydes (17). In particular, Akr1b8 displays the most efficient phospholipid aldehyde and HNEreductase activity in mouse tissues (17, 28, 37) (**Figure 3**; **Table 4**). All these enzymatic data suggest that in the adrenal cortex, Akr1b8 isoform could be devoted to detoxify aldehyde lipids abundantly accumulated in this tissue (38). Furthermore, expression of an antisense RNA directed against *Akr1b7* suppressed isocaproaldehyde reductase activity in adrenocortical Y1 cells without any alteration of Akr1b8 protein accumulation (29). In view of its enzymatic features and constitutive expression, Akr1b8 is unlikely to be the principal isocaproaldehyde reductase in the adrenocortical gland (28). *Akr1b8* gene disruption in mice led to reduced lipid synthesis and diminished proliferation of colonic epithelial cells but had no evident effect on general appearance, body weight, and reproduction. However, *in vivo* adrenal Akr1b8 physiological role remains to be examined since *Akr1b8* gene disruption first report did not notice evident effect on the adrenal physiology (39).

Whether *Akr1b8* and human *AKR1B10* gene can be considered as ortholog is still a matter of debate since they share high sequence identity, and proteins display several close structural and enzymatic properties (11) (**Tables 1** and **4**). In contrast to *Akr1b8*, *AKR1B10* gene expression is not controlled by FGF (12). Although *AKR1B10* mRNA was initially detected in adrenal glands using a human RNA Master Blot, to date, there is no more information available on its *in situ* localization and transcriptional control in this organ (1). Moreover, *AKR1B10* gene expression pattern only partially overlaps that of *Akr1b8*, since *AKR1B10* transcripts are absent from heart, lung, or testis (7, 11).

Comparative studies demonstrated that AKR1B10 exhibits higher 4-HNE-reductase activity than AKR1B1, while lower than the murine Akr1b8 (11, 16). *Ex vivo* studies revealed that both human AKR1B1 and -B10 also share the ability to reduce isocaproaldehyde (1, 40). Nevertheless, in a comparative enzymatic study, Hara and colleagues showed that AKR1B1 had a more effective isocaproaldehyde reductase activity than AKR1B10, suggesting that in human steroidogenic organs, the latter was unlikely to play a major role in the detoxification of steroidogenic by-products (41).

The AKR superfamily has been added as a novel group of cytosolic enzymes that could contribute to retinoid–redox conversion. Based on their cofactor specificity (NADPH), AKR work in the reductive direction (42). Retinol (vitamin A) and its derivatives, retinaldehyde and retinoic acid (RA), are essential for the growth and maintenance of many tissues. RA is a key molecule in the development of different vertebrate organs by promoting cell differentiation and apoptosis. The control of retinaldehyde levels is essential in the regulation of RA synthesis and therefore of its signaling role. Once synthesized from β-carotene through the β-carotene 15,15′ monooxygenase 1 (BCO1), retinaldehyde has two alternative fates, its irreversible oxidation to RA (metabolism fate) by the aldehyde dehydrogenases (ADH) or its reduction back to retinol (storage fate) by the retinaldehyde reductase activity of AKR (43) (**Figure 3B**; **Table 4**). Comparative *in vitro* enzymatic studies on murine and human AKRs have fairly evidenced that among AKR1B proteins, AKR1B10 is so far the only retinaldehyde reductase with the highest *k*cat value for the retinaldehyde reduction (11, 12, 44). *Ex vivo* AKR1B10 overexpression in different cell systems demonstrated its contribution in increasing retinol production (45, 46). In rodent, previous data evidenced that normal adrenal gland may function as an important site of retinoic acid synthesis involving class I- and IV-ADH, thus furthering retinaldehyde metabolism rather than its storage through the AKR activity (47). According to its expression, whether the well-established retinaldehyde reductase activity of AKR1B10 is operated in normal human adrenal physiology remains to be explored.

*AKR1B10* expression was initially characterized in hepatocellular carcinoma and subsequently found to be altered by tumorigenesis process in several other organs (7, 48–50). Moreover, *AKR1B10* expression was associated with smoker's non-small cell lung carcinomas (48) and was suggested to be involved in drug resistance (51). A putative mechanism by which the activity of AKR1B enzymes could promote tumor growth is the conversion of retinaldehyde to retinol resulting in RA deprivation and blockage of its differentiating effect, promoting cell proliferation and fostering tumorigenesis (43). Furthermore, recent studies have shown that in breast cancer cells, AKR1B10 associates with acetyl-CoA carboxylase-alpha (ACCA), the rate-limiting enzyme of *de novo* synthesis of long-chain fatty acids, and blocks its ubiquitination and proteasome degradation. Long-chain fatty acids are the building blocks of biomembranes and the precursor of lipid second messengers, playing a critical role in cell growth and proliferation (52). The AKR1B10-mediated regulation on ACCA stability represents a novel regulatory mechanism, in which AKR1B10 promotes cell survival *via* modulating lipid synthesis, mitochondrial function, oxidative stress, and carbonyl levels (53).

Adrenocortical carcinomas are very aggressive and rare malignant tumors with poor prognosis (54). Microarray analysis was used to seek molecular predictors of malignancy and survival in a large cohort of unilateral adrenocortical tumors (http://www.ebi.ac.uk/arrayexpress, experiment E-TABM-311). Unsupervised clustering analysis allowed robust discrimination of malignant and benign tumors. On the basis of this analysis, *AKR1B10* expression was not found to be associated with the ACC group (35).

### Akr1b7: Expression Profile, Detoxification Function, and Paracrine Action

High levels of *Akr1b7* transcripts were initially observed by *in situ* hybridization in fetal and adult murine adrenal cortex but were undetectable in the medulla (2). We confirmed these results by immunohistochemistry experiments, which allowed us to further restrict major Akr1b7 expression to the *zona fasciculata* (55). *In vivo*, ACTH suppression with dexamethasone treatment resulted in a marked decrease of *Akr1b7* mRNA levels that were restored when the treated mice were injected with exogenous ACTH. This ACTH/cAMP-induced *Akr1b7* transcription was blocked by a PKA inhibitor (H89) in the murine adrenocortical ATC and Y1 cell lines (55, 56).

In the adrenal gland, basal and ACTH-induced expressions of *Akr1b7* gene depend on three SF1 response element (SFRE) and on other *cis*-elements located in the upstream promoter region (**Figure 3**). Using transgenic mice and transfection experiments, we characterized a cryptic SFRE 102 bp upstream of the transcription start site that supports basal expression of *Akr1b7* in the adrenal cortex. Among the two other SFREs identified further upstream, the site at −458 was a *bona fide* SFRE, essential for both basal and cAMP-stimulated promoter activity. The last SFRE, an Sp1, and C/EBPβ binding sites, respectively, localized at positions −503, −52, and −61 are all involved in cAMP responsiveness (57, 58).

AKR1Bs are capable to handle the large amount of isocaproaldehyde, a toxic by-product coming from the cholesterol side-chain cleavage during the initial step of steroid biosynthesis. Furthermore, isocaproaldehyde accumulation decreased viability of Y1 cells (29). Although Akr1b3, Akr1b7, and Akr1b8 all were able to reduce isocaproaldehyde, the two former seemed to be the more efficient reductases for this substrate (28). However, the silencing of *Akr1b7* gene was sufficient to abolish the cAMP-induced isocaproaldehyde reductase activity in Y1 cells. Therefore, Akr1b7 was the main enzyme in charge of isocaproaldehyde detoxification in the adrenal cortex (29). Altogether, these data showed that in the adrenal cortex, ACTH not only controls expression of enzymes synthetizing steroids but also of proteins scavenging toxic by-products derived from steroidogenesis.

Madore et al. established that the bovine 20α-hydroxysteroid dehydrogenase, AKR1B5 was responsible for PGF2α synthesis in the endometrium (59). Thereafter, we demonstrated by *ex vivo* studies, that AKR1B1, Akr1b3, and Akr1b7 were also able to reduce PGH2 into PGF2α, whereas Akr1b8 and AKR1B10 were devoid of this PGF2α synthase activity (**Table 4**). Due to their recent discovery, this 9-,11-endoperoxide reductase activity has not been investigated yet for Akr1b16 and AKR1B15. Moreover, their enzymatic constants suggested that AKR1B1, Akr1b3, and Akr1b7 had a higher 9-,11-endoperoxide reductase activity than the other PGF synthases already described (34). Prostaglandins are paracrine/autocrine signal molecules produced from a common precursor, PGH2, which is derived from arachidonic acid by COX-1 or COX-2. Unlike COX-1, which is a constitutively expressed enzyme, COX-2 is not expressed in most organs under basal conditions but can be stimulated by inflammation and various mitogenic factors (60). Following these observations, we carefully examined the PGF2α biosynthetic pathway in the adrenal gland (27).

Prostaglandin F2α was produced by both cortical (steroidogenic cells) and medullary (chromaffin cells) tissue of the adrenal gland. In primary adrenocortical cell culture, PGF2α release was induced 2.5-fold by ACTH treatment. This secretion was correlated with ACTH responsiveness of both COX-2 and Akr1b7. Using *ex vivo* gain- and loss-of-function strategies, we demonstrated the pivotal role of Akr1b7 in ACTH-induced PGF2α release, and it is functionally coupled with COX-2. In the adrenal medulla in which *Akr1b7* was not expressed, PGF2α was produced from the coordinated activities of Akr1b3 and COX-1. Adrenal expression of PGF2α-specific receptor (FP) was restricted to the chromaffin cells, suggesting that both autocrine and paracrine mechanisms (within the medulla and between steroidogenic and medulla cells, respectively) were relaying PGF2α action. In agreement with this hypothesis, we demonstrated that PGF2α repressed both basal and glucocorticoid-induced dopamine release in the chromaffin cell line MPC862L. Comparison of the PGF2α-responsiveness of isolated cells and whole adrenal tissue cultures showed that PGF2α-mediated repression of glucocorticoid release is an indirect mechanism relying on a decrease in catecholamine secretion, which in turn decreased cortical steroidogenesis.

These functional data led us to propose an intra-adrenal feedback loop in which adrenal endocrine activities are regulated through the involvement of AKRs [**Figure 4** and Ref. (18)]. Surprisingly, however, the absence of Akr1b7 *in vivo* did not

FIGURE 4 | Proposed model integrating dual functions of aldose reductases in the regulation of mouse adrenal endocrine functions [adapted from Ref. (18)]. AKR1B family is endowed with enzymatic activities that at least ensure two metabolic functions: the production of PGF2α and the detoxification of lipid aldehydes. Arachidonic acid (AA) is metabolized into PGH2, the precursor of all prostanoids, by COX enzymes and then converted into PGF2α by the 9-,11-endoperoxyde reductase activity of PGF synthases of the AKR1B family. AKR1B are also capable to reduce toxic lipid aldehydes resulting from the particularly high prooxidant activities of P450 cytochromes in adrenocortical cells (i.e., isocaproaldehyde and 4-HNE). The mechanism integrating these dual functions was the following: (1) In basal conditions, PGF2α is constitutively secreted by chromaffin cells (by the coupling of COX-1 and Akr1b3), thus regulating catecholamine production and also limiting their paracrine action on steroidogenesis. (2) During a stress situation, ACTH transiently induces COX-2 and Akr1b7 expression, which results in PGF2α production inside the cortex. PGF2α produced in the cortex then represses catecholamine release by the medulla *via* a paracrine action on its FP receptor. Decreased catecholamine release in turn reduces the effect of ACTH on glucocorticoids production (27). After the stress response has ended, COX-2 returns to undetectable levels. The coupling between Akr1b7 and COX-2 does not take place. Then, Akr1b7 together with Akr1b8 and Akr1b3 function only as cortical detoxifying enzymes of the harmful aldehydes produced under chronic/basal stimulation of steroidogenesis. Catecho, catecholamine; Glucocort, glucocorticoids.

affect basal adrenocortical function as illustrated by normal glucocorticoid plasma levels in *Akr1b7*−*/*− mice (61). Indeed, these mice displayed an obese phenotype that did not rely on adrenal dysfunction but on the lack of Akr1b7-dependent production of PGF2α within the stromal vascular adipose tissue (3, 61). Adrenal expression of Akr1b3 and b8 is not affected in knockout mice and since they all share redundant enzymatic activities regarding detoxification of lipid aldehydes (see Akr1b3/AKR1B1: Expression Pattern and Relevant Functions and Akr1b8/ AKR1B10: Phylogeny, Regulation, and Enzymatic Specificities), the remaining isoforms can compensate the absence of Akr1b7 at least in basal conditions. Importantly, Akr1b7 is the only one out of the three adrenal isoforms to be ACTH-responsive (27) and also the most abundantly expressed (3). Taken together, these hallmarks would predict that physiological importance of Akr1b enzymes in adrenal function should be rather explored under stress conditions during which scavenging capacity of constitutive (and less abundant) isoforms should be exhausted.

### FUTURE DIRECTIONS

Fighting against oxidative stress is a challenging but mandatory task for adrenocortical cells. Indeed, P450 cytochrome systems involved in steroidogenesis, and in particular glucocorticoid production, contribute very significantly to oxidative stress by cellular reactive oxygen species (ROS) production (62). The redox imbalance due to excessive ROS production can cause adrenal damage that may progress to severe insufficiency, including familial glucocorticoid deficiency (FGD). Therefore, adrenal cortex is well supplied in antioxidant defense genes encoding enzymes of the superoxide dismutase (SOD), glutathione peroxidase (GPX), and peroxiredoxin (PRDX) families

### REFERENCES

[for review, see Ref. (63)]. Since the precursor works of Feige's group showing the ACTH responsiveness of *SOD2* expression, the expected coordinated regulation of antioxidant enzymatic systems and P450s systems producing prooxidant by-products has been somewhat neglected (64). Accordingly, AKR1B enzymes family may be considered as antioxidant defense genes. Among these, ACTH-responsive ones, e.g., *Akr1b7* and *SOD2* genes, could participate in the adaptive response of antioxidant systems of adrenal cortex under stress conditions. Disturbance in redox homeostasis was the most recently discovered cause of FGD and mutations in *NNT* gene (nicotinamide nucleotide transhydrogenase) account for about 10% of cases (65). NNT ensures mitochondrial NADPH supply that is essential to ROS detoxification enzymatic systems. Then, it would be interesting to know whether adrenal-specific deficit in AKR1B enzymes could contribute to cortical damage or adrenal insufficiency in mice carrying a spontaneous *Nnt* mutation (66). This could provide the proof of principle for studying the physiological contribution of AKR1B family in detoxifying function in steroidogenic organs and beyond.

### AUTHOR CONTRIBUTIONS

All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.

### FUNDING

This work was supported by funds from the Centre National de la Recherche Scientifique, Clermont Université, Institut National de la Santé et de la Recherche Médicale and grant from the Région Auvergne.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Pastel, Pointud, Martinez and Lefrançois-Martinez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Adrenocortical Gap Junctions and Their Functions

### *Cheryl L. Bell and Sandra A. Murray\**

*Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA*

Adrenal cortical steroidogenesis and proliferation are thought to be modulated by gap junction-mediated direct cell–cell communication of regulatory molecules between cells. Such communication is regulated by the number of gap junction channels between contacting cells, the rate at which information flows between these channels, and the rate of channel turnover. Knowledge of the factors regulating gap junction-mediated communication and the turnover process are critical to an understanding of adrenal cortical cell functions, including development, hormonal response to adrenocorticotropin, and neoplastic dedifferentiation. Here, we review what is known about gap junctions in the adrenal gland, with particular attention to their role in adrenocortical cell steroidogenesis and proliferation. Information and insight gained from electrophysiological, molecular biological, and imaging (immunocytochemical, freeze fracture, transmission electron microscopic, and live cell) techniques will be provided.

#### *Edited by:*

*Nicole Gallo-Payet, University of Sherbrooke, Canada*

#### *Reviewed by:*

*Vance Trudeau, University of Ottawa, Canada Trond Aasen, Vall d'Hebron Research Institute (VHIR), Spain*

#### *\*Correspondence:*

*Sandra A. Murray<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology*

*Received: 04 May 2016 Accepted: 20 June 2016 Published: 29 June 2016*

#### *Citation:*

*Bell CL and Murray SA (2016) Adrenocortical Gap Junctions and Their Functions. Front. Endocrinol. 7:82. doi: 10.3389/fendo.2016.00082*

Keywords: ACTH, connexin, gap junction plaques, gap junction vesicles, steroidogenesis

### INTRODUCTION

The adrenal is a complex gland that is histologically and functionally two tissues, the cortex and medulla, within a connective tissue capsule (1). Not only the two regions of the adrenal gland are from different embryonic origins but also the cells of the medulla are composed of cells that have a neuroendocrine function, while the cells of the cortex are epithelial cells that function in endocrine metabolism. There is evidence that cells of both the cortex and the medulla are regulated by cell–cell communication of regulatory molecules through membrane channels, called gap junctions (2–8). The efficiency of the adrenal gland, as well as other endocrine glands, to respond to stimulation is thought to depend not only on hormone receptor interaction but also on intercellular communication through gap junctions.

Gap junctions in the adrenal gland, as in other tissues, provide low-resistance pathways for the direct intercellular exchange of small molecules (9, 10). In early years, gap junctions and cell communication were mainly studied with electron microscopic (11–13), electrophysiological (14), and fluorescent dye transfer (15, 16) techniques. More recently, however, the proteins (connexins) composing the gap junction pore have been identified, and the tissue distribution of the different connexin family members has been demonstrated (17–20). Further, the molecular details of the

**Abbreviations:** 18-alpha/beta GA, 18-alpha/beta glycerrhetinic acid; ACTH, adrenocorticotropin hormone; DbcAMP, dibutyryl cyclic adenosine monophosphate; FSH, follicle-stimulating hormone; GFP, green fluorescent protein; LAMP, lysosomal-associated membrane protein; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; PKC, protein kinase C; RNAi, RNA interference; SBAC, bovine adrenal cortical cell line; SW-13, Scott white human adrenal cortical tumor cell line; TEM, transmission electron microscopy; TPA, 12-*O*-tetradecanoylphorbol-13-acetate; Y-1, mouse adrenal cortical tumor cell line.

assembly of connexins into functional gap junction channels, the involvement of kinases in this assembly, and the architectural arrangement of connexins into functional pore complexes have been described (21, 22).

In this review, we present a historical summary of gap junctions in the adrenal cortex from their discovery with imaging and electrophysiological techniques to current studies of the connexin types, distribution, abundance, and turnover. The role of gap junctions in the adrenal cortical response to adrenocorticotropin (ACTH) will be discussed, and the fundamental concepts and implication of gap junctions in steroidogenesis, proliferation, and cancer will be analyzed. Finally, gap junctionmediated cross talk between the cells of the adrenal cortex and medulla will be discussed, as it relates to adrenocortical function. We will begin with a brief review of gap junctions in the adrenal cortex.

### CHARACTERIZATION OF GAP JUNCTIONS IN THE ADRENAL CORTEX

Gap junctions occur between the membranes of two closely opposed cells and are characterized by the pairing of intramembranous connexin complexes across a 2–4 nm gap (**Figures 1** and **2**) (23). The first transmission electron microscopic images of gap junction plaques in the adrenal gland were acquired from fetal rat adrenals in 1970 (24) and more extensively from adult glands of a number of animals in 1972 (11, 25). Gap junctions in the adrenal resembled those of other tissues and were positively identified by the presence of the characteristic pentalaminar membrane and the 2–4 nm gap separating the membrane of two

adjacent cells (**Figure 2A**). The area of membrane covered by gap junction plaques (plaque size) and the packing pattern of adrenal cortical cell gap junction channels could be obtained with freeze-fracture electron microscopy (11). With this technique, the adrenal cell membrane bilayer was split in the hydrophobic plane (26), and the typical protoplasmic (P)-fracture face and extracellular (E)-fracture face distribution of membrane particles was used to identify gap junctions (**Figure 2B**). Gap junctions could be distinguished by the observed clustering of 8.5 nm particles on the P-fracture face and pits on the E-fracture face (27, 28) (**Figure 2B**). With freeze-fracture techniques, the size of gap junction plaques was demonstrated in the rat adrenal cortex to be larger and more abundant than those found in most other tissues of the body, especially those plaques in the area of the adrenal cortex near or juxtaposed to the medulla (24). Gap junction plaques have now been described in the adrenal from a vast number of different mammalian species (6, 29–31). An important advantage of freeze-fracture imaging was the capacity to confirm that the adrenal cortical gap junctions were composed of aggregates of intramembranous particles that were paired with one another across the intercellular space. The observation that these paired particles extended across the gap, seen with transmission electron microscopy, provided structural evidence for the existence of channels, which could serve as means for movement of molecules between cells. However, the critical role of gap junctions as channels for communication was established by experiments in which it was demonstrated that hepatocytes and myocardial cells only passed current if gap junctions were observed and if gap junctions were not present, current did not pass between cells (32).

The movement of current was first demonstrated in the adrenal cortex with electrophysiological techniques in experiments, which used adult and fetal rabbit adrenal gland slices (10). Although such studies confirmed that material was capable of communicating between cells in the adrenal, the function of this movement was not addressed in these early studies. In addition, information on the possible size of molecules that could move between the gap junction channels was provided from dye transfer studies in which molecules smaller than 1,000 Da could communicate between cells while larger molecules were excluded (16, 33, 34). Based mainly on the early electrophysiological and dye transfer studies, as well as transmission electron microscopic and freeze-fracture images, it was hypothesized that gap junctions allowed the passage of molecules between adjacent cells, and thus, modulate adrenal cortical cell population growth and hormonal response (8, 35). However, the molecules composing and regulating the gap junction pore and, more important, the definitive role of gap junction-mediated communication in adrenal cortical function remained to be defined.

Our knowledge concerning the role of gap junctions, in general, was greatly enhanced by the isolation and characterization of the connexin proteins that composed gap junction pores. It has become clear that the particles seen with freeze-fracture electron microscopy are the gap junction channels and that each gap junction channel consists of 12 connexin molecules, 6 from one cell docked to 6 in the adjacent cell. The clustering of these channels forms the gap junction plaque (27, 36).

The production of antibodies directed at the connexin proteins allowed the detection of gap junction protein types with immunofluorescence and western blot techniques (19, 20, 37). In humans, there are 21 different connexin types that differ by their amino acid sequences and molecular weights (38). Connexin 43 (Cx43) gap junction protein was demonstrated as the major, if not only, connexin gap junction protein type in the adrenal cortex (5, 30, 31, 39, 40). While investigators have also reported Cx26, Cx32, and Cx50 in humans (41), their expression has not been reported in other mammals (3, 6, 7, 42). It should be noted, however, that of the 21 known connexin family members (38), only 6 have been extensively evaluated in the adrenal cortex, and it is possible that others will be detected with further analysis.

With immunofluorescence microscopy, the distribution of Cx43 could be quickly detected and its distribution more reliably compared with adrenal zones than with conventional transmission electron microscopy (5, 30, 40, 43). The Cx43 gap junctions in the adrenal cortex appear as small puncta or longer plaques on the cell surface between contacting cells (**Figures 2C,D**). The cells of the adrenal cortex are polyhedral in shape, and their three-dimensional relationship to one another has been revealed with serial section transmission electron microscopy (44) and scanning electron microscopy of freeze-cracked adrenal glands (45). Based on the three-dimensional imaging provided by these techniques, gap junctions are thought to form at smooth "contact" sites and on facets on microvilli of cells (45). The facets from two cells were observed to be in close apposition, and it is here as well as at the smooth sites on the cell body that the gap junctions most likely are formed (45).

In addition to the typical surface gap junction plaques, cytoplasmic gap junction vesicles have also been reported in the adrenal gland and in adrenal cell cultures with both transmission electron microscopic and immunocytochemical techniques (46–49). It has been confirmed with live cell imaging of cells expressing green fluorescent protein construct (Cx43-GFP) that these annular gap junction vesicles form from a unique process in which the gap junction membrane of one cell is internalized into the cytoplasm of the adjacent cell to form a double-membraned vesicle composed of gap junction protein. This internalization process occurs from the central regions of the gap junction plaques or, in some cases, the entire gap junction plaque membrane is internalized. Once internalized, the annular gap junction vesicle is degraded (50–52), and thus, this is a method for disassembling gap junction plaques and regulating communication. In contrast, gap junction plaque assembly occurs by the addition of new gap junction channels at the gap junction plaque periphery (53). Mechanisms for controlling cell–cell communication are thought to involve both the assembly of gap junction plaques at the cell surface and the disassembly of these plaques by an internalization process that results in annular gap junction vesicle release into the cytoplasm. The capacity to specifically analyze connexin protein distribution within specific compartments is particularly critical for the study of tissues, such as the adrenal, which have cells that express different steroidogenic enzymes and respond differently to stimuli, based on their specific zonal locations.

### GAP JUNCTION DISTRIBUTION IN THE ADRENAL CORTEX

In the human, the adrenal cortex can be divided into three morphologically and functionally distinct zones: the outer most zone, zona glomerulosa, and the inner zones, zona fasciculata (ZF) and zona reticularis (ZR). These zones are composed of cells that express different steroidogenic enzymes and thus produce different steroid hormones. Specifically, the ZF produces glucocorticoids, and the ZR produces androgens. Both of these inner zones secrete hormones in response to ACTH. The outer zone, however, produces aldosterone in response to changes in sodium, potassium, and the peptide hormone, angiotensin II.

Just as the three zones of the adrenal have been demonstrated to be morphologically and functionally distinct, the level of expression of gap junction protein in the three zones of the adrenal has been demonstrated to differ using immunocytochemistry (**Figures 3** and **4**).  Specifically, little or no Cx43 gap junction protein was detected between adrenal cells in the zona glomerulosa. In contrast, numerous gap junction plaques were evident at areas of cell–cell contact in the inner cortical areas, such as the ZF and ZR (**Figure 3**). The distribution of gap junctions once speculated from transmission electron and freeze-fracture microscopy (29, 55) could be confirmed and, more importantly quantitated, while simultaneously validating which zone was being viewed. With computer-assisted microspectrofluorometric image analysis, it was demonstrated that there were twice as many gap junctions per area between cells in the ZR than between cells in the ZF (6). While the number of gap junctions differed, there were no significant differences in the average size of gap junctions in the rat ZF compared with those in the ZR.

Consistent with the immunocytochemical quantitative analysis, when the expression of Cx43-encoded mRNA was analyzed with Northern Blot techniques in samples prepared from the capsule of the adrenal gland, which contain adherent zona glomerulosa cells, there was little gap junction RNA detected. In contrast, samples prepared from decapsulated glands, which contained mainly the ZF/ZR cells, had an abundance of gap junction RNA. It is thought that the detection of small amounts of Cx43 encoded mRNA in the zona glomerulosa with Northern Blot and almost no Cx43 gap junction proteins detected with immunocytochemical analysis may reflect possible contamination of the zona glomerulosa-enriched samples with cells from the ZF. In addition, some of the mRNA Cx43 detected with Northern Blot in the zona glomerulosa-enriched samples is thought to come from the presence of fibroblastic cells from the connective tissue capsule and trabeculea that project into the substance of the cortex in the sample (**Figure 3D**). In fact, these trabeculea within the substance of the gland were revealed with immunocytochemistry. To increase the accuracy for comparing the distribution of gap junctions with adrenal cortical zones, Oil Red O staining, which detects lipid droplets in the cytoplasm of cells in the ZF and ZR (56), has been used. The Oil Red O stain is much less abundant in zona glomerulosa cells that have few, if any, lipid droplets (56).

FIGURE 3 | Gap junction (Cx43) localization and dye communication in the intact adrenal gland. Immunohistochemical localization of Cx43 gap junction proteins revealed extensive staining in the zona fasciculata (ZF) and zona reticularis (ZR), while there was limited staining in the zona glomerulosa (ZF) [left panels: (A,C)]. Correspondingly, lucifer yellow dye communication between cells was more abundant in the inner zones of the adrenal cortex (ACTH responsive areas) than in the outer zone [right panels: (B,D)]. Capsule (arrowheads), connective tissue trabecule (arrow), and Cx43 (white puncta). Bars: (A,B) 50 μm and (C,D) 30 μm [modified from Ref. (30)].

The pattern of Oil Red O staining distinguished the cells of the zona glomerulosa from cells of ZF and ZR and substantiated that the cells which lack Cx43 gap junction staining are indeed cells of the zona glomerulosa (**Figure 4**) (30).

In parallel to the differential localization of gap junction plaques is the pattern of dye communication in the intact adrenal cortex. Specifically, lucifer yellow dye moved extensively in the inner cortex while dye communication was limited to the capsular projections of connective tissue between the cells of the zona glomerulosa (**Figures 3B,D** and **4B,D**) (5, 30). Dye communication was not observed between cells of the zona glomerulosa. This lack of dye movement would be consistent with the observed absence of Cx43 protein in immunofluorescence studies and would further support the suggestion that other connexin family members do not assemble into functional gap junctions in this zone. In the inner two cortical zones, where gap junctions were plentiful, dye was transferred (30). It should be noted that while Cx43 was found in the inner two zones, it is possible that connexin types, other than Cx43, may be located

and nuclei. Bars: (A–D) 30 μm [modified from Ref. (30)].

in these inner two zones since not all of the 21 known connexin family members have been evaluated. In addition, there is a possibility that other, yet to be discovered, connexin type could be detected in the future. It is speculated that while cells of the zona glomerulosa, for reasons yet to be determined, are less dependent on cell–cell communication, the communication of a regulatory molecule between cells of the inner two layers modulates their functions.

### ROLE OF GAP JUNCTIONS IN ADRENAL CORTICAL FUNCTION

### Steroidogenesis

Gap junctions, for years, have been suggested to play a key role in a number of physiological phenomena (3, 57, 58). However, in early years, this proposed role was solely based on the observations that dye and current could move between cells that had gap junctions and not on experimental evidence that demonstrates actual physiological responses. Adrenal cortical cells were one of the first endocrine cell types in which a direct relationship was established between gap junctions and physiological responses (59). Furthermore, the findings in the adrenal cortex have been used to lend support to the speculations that gap junctions influence physiological events in other cell types (60, 61).

The first set of experiments, which demonstrated functional gap junction-mediated communication, were studies in which adrenal cells were placed into culture with ovarian granulosa cells. It is well documented that cells of the inner adrenal cortical zones possess specific receptors for ACTH and, when bound to its receptor, ACTH elicits a number of responses (62, 63), including an activation of cAMP-dependent protein kinase A (PKA) (59, 64), an increase in steroid synthesis (7, 65, 66), and a decrease in proliferation of cells in culture (2, 8, 67). In the adrenal/granulosa coculture populations, a cytochemical method was used to specifically localize free catalytic subunits (C) of PKA at subcellular, light microscopic levels of resolution (59, 68). In these studies, heterotypic, adrenal/granulosa, cell pairs were demonstrated to form gap junctions and ACTH treatment activated PKA in the adrenal cell populations (59). Moreover, ACTH stimulation not only, as expected, activated PKA in the adrenal cells but also with time, within the granulosa cells that were in contact with adrenal cells. The ovarian granulosa cells lacked ACTH receptors but had receptors for follicle-stimulating hormone (FSH), which activates PKA (69). Treatment of the coculture cell population with FSH activated PKA in the granulosa cells and in the adrenal cells that formed cell contacts with granulosa cells (**Figure 5**). Importantly, adrenal cell secretion of steroid hormones was increased (59). The finding that hormone treatment stimulated bidirectional exchange of a signal that initiated PKA activation in adrenal/granulosa coculture cell populations provided compelling evidence that the hormone-induced intercellular communication, which activated PKA, could serve a biological role.

In further support of the biological role of gap junctions in adrenal cortical function, inhibition of gap-junctional communication with 18-alpha glycyrrhetinic acid (18-alpha GA) treatment decreased the steroidogenic responsiveness of bovine adrenal cells in culture to ACTH (4). Specifically, it was demonstrated that 18-alpha GA treatment decreased the steroidogenic response to a submaximal dose of ACTH but did not decrease steroidogenesis if cells were treated with saturating doses of ACTH (4). A similar decrease in ACTH-stimulated steroidogenesis was demonstrated in bovine and human cell lines and in rat primary cell populations treated with 18-alpha GA (8). The effects of treatment with this glycyrrhetinic acid derivative were also substantiated in experiments in which Cx43 antisense transfection techniques were used to inhibit gap junction-mediated communication (8). Cell populations expressing Cx43 antisense grew faster, had decreased capacity for cell–cell communication, and had a diminished steroidogenic responsiveness to ACTH treatment compared with null transfected control populations (8). This decrease in ACTH-mediated steroidogenesis following gap junction inhibition by either chemical treatment or Cx43 antisense expression is consistent with the concept that gap junction-mediated communication is a necessary factor in hormonal response. It has been suggested that gap junction communication of cAMP signaling, and subsequent PKA activation in the recipient cell, may increase the efficiency of hormone response by facilitating amplification of ACTH signaling throughout the population. This would be particularly useful at submaximal stimulation doses of the hormone, as suggested by Munari-Silem, when only a small population of the cells may be responding to ACTH (4).

In addition to 18-alpha, 18-beta glycerrhetinic acid (18-beta GA) has also been used in a number of studies to inhibit gap junction communication (70–72). Huang and colleagues found that treatment with 18-beta GA increased the basal levels of steroidogenesis secreted in rat primary adrenal cell cultures (73). These findings are interesting given that the increased steroidogenesis appears to be PKA-independent and would suggest a direct effect of the 18-beta GA on the production of steroidogenic enzymes in addition to their effect on inhibition of gap junction-mediated cell communication. It should be noted that neither cell communication nor the effect of ACTH stimulation was measured in this study. Further investigations are needed to determine if the concentrations of 18-beta GA used in this study eliminated cell–cell communication, and more importantly, if the ACTH-stimulated steroidogenesis at submaximal and saturating treatment doses were altered. It should also be noted that both 18-alpha and 18-beta GA have formulas very similar to that of cortisol and may be mistaken, with some assay methods, for an adrenal steroid (74).

### Proliferation

In the intact adrenal gland, it was noted that the cells of the zona glomerulosa, which had few if any gap junctions, divided rapidly. In contrast, cells of the inner zones, where gap junctions were more abundant, proliferated more slowly (6, 40). Based on this inverse relationship between the rate of proliferation in the adrenal zones and the presence of gap junctions, it was suggested that gap junctions play a role in regulating cell proliferation in the adrenal cortex.

In the zona glomerulosa, it has been suggested that the lack of gap junctions and thus capacity for direct communication of growth regulatory molecules would result in more cell proliferation in this zone. Along these same lines of evidence, the slower proliferation rate of cells in the inner zones, where gap junctions are more abundant (5, 40), would be consistent with their greater capacity to communicate growth inhibitory signals. Many factors are known to regulate growth, and gap junction-mediated communication may play a key role by facilitating the movement of these factors between the cells.

In support of the hypothesis that gap junctions contribute to the control of adrenal cell proliferation, possibly by allowing passage of molecules between cells, are the findings that cell proliferation of bovine adrenal cortical cell (SBAC) populations was significantly increased if gap junction protein expression was suppressed by Cx43 gap junction cDNA antisense transfection techniques (8). The average number and size of gap junction plaques decreased while the rate of population growth increased in these studies (8). Conversely, when adrenal cells were treated with ACTH, which increased gap junction protein expression, there was a corresponding decrease in cell population growth (8). Both the ACTH-stimulated increase in gap junction protein expression and decrease in cell population growth were mimicked by treatment with the second messenger, dibutyryl cyclic adenosine monophosphate [DbcAMP (1 mM)]. It is, thus, thought that the ACTH-induced alterations in Cx43 gap junction protein expression and proliferation are modulated by a PKA mechanism. Further, the findings of an inverse relationship between gap junctions and proliferation would suggest that, in the adrenal cortex, along with the multiple mechanisms known to be involved in controlling adrenal gland structure and function, ACTH-stimulated intercellular communication *via* gap junctions may represent an important factor in adrenal gland behavior. However, not only is gap junction-mediated cell communication thought to regulate ACTH-induced changes in steroidogenesis and proliferation but also there is compelling evidence that ACTH serves to regulate gap junction protein expression and stability at the cell surface.

### REGULATION OF GAP JUNCTION EXPRESSION

In a number of studies of adrenal cells in culture, ACTH has been demonstrated to increase gap junction protein expression and to increase the size and number of surface gap junction plaques (4, 7, 8). These effects on gap junctions can be mimicked by treatments that increase cAMP levels and activate PKA (59, 64). Specifically, an increase in gap junction plaque size and number at the cell surface as well as the decrease in gap junction plaque disassembly (internalization to form annular gap junctions) was reported following DbcAMP treatment. These results are consistent with the theory that the changes in gap junctions following ACTH treatment were dependent on cAMP and the activation of protein kinase. Conversely, elevation of cAMP and PKA activation has been demonstrated to decrease the number of annular gap junctions in adrenal cortical cells (75). This suggests that PKA activation may decrease gap junction plaque internalization, which would further contribute to the observed increase in gap junction plaques at the cell surface following treatments that elevate cAMP levels in adrenal cell populations.

In addition to the findings made in adrenal cell cultures, the relationship between gap junction protein expression, occurrence, distribution, and ACTH levels in the body have been evaluated in studies in which the tropic state of the adrenal gland was altered by surgical removal of the pituitary (46). The removal of the pituitary, termed hypophysectomy, eliminates the source of ACTH since the pituitary secretes this as well as a variety of other hormones that are either produced by cells of the anterior pituitary (growth hormone, gonadotrophins, prolactin, and thyroid stimulating hormone) or by cells of the hypothalamus and then stored in the pituitary (antidiuretic hormone and oxytocin) (1, 76). The elimination of ACTH by hypophysectomy led to a profound atrophy of the cortex, which was more marked in the inner zones (ZF and ZR) than in the zona glomerulosa (46). While increasing ACTH levels increased gap junctions, eliminating ACTH by perturbing the pituitary–adrenal gland axis by hypophysectomy in mice led to diminished Cx43 gap junction expression mainly in the ZF (**Figure 6**) (31). If these hypophysectomized animals were treated with ACTH, Cx43 gap junction plaque size was increased (46). The increase in gap junction protein expression occurred in the ACTH-dependent zones (ZF and ZR) with no change in the ACTH-independent zone (zona glomerulosa). Thus Cx43 gap junction protein expression can be regulated *in vivo* as well as in adrenal cell populations maintained in culture, in further support that ACTH can modulate gap junctions.

The molecular mechanism for how ACTH may modulate gap junctions has not been demonstrated in the adrenal gland. It is well documented, however, that ACTH treatment results in PKA activation (64); reviewed by Ruggiero and Lalli (77), and in a number of other cell types, activation of PKA has been shown to increase gap junction plaque assembly by facilitating the phosphorylation of gap junction protein (78). Specifically, PKA activation results in the phosphorylation of serines (S364, S365,

FIGURE 6 | Immunohistochemical demonstration of gap junction (Cx43) distribution in the adrenal gland. Gap junction proteins were abundant in the inner cortex of the mouse adrenal (A). Hypophysectomy led to diminished Cx43 gap junction expression mainly in the zona fasciculata at 33 days post-surgery (B) [modified from Ref. (31)].

S369, and S373) on the C-terminal tail of Cx43 (78). Treatment with ACTH, based on this information, would be expected to phosphorylate one or more of these serines. There is a need, however, for increased information on the relationship between ACTH treatment and connexin phosphorylation if we are to fully understand the role of gap junction function in adrenal cell behavior.

To date, the largest increase in information on gap junction regulation and function comes from the knowledge of which Cx43 C-terminal tail amino acids are phosphorylated and dephosphorylated (22, 78–81). Phosphorylation/dephosphorylation events on the C-terminal tail of Cx43 are dependent on activation of PKA, as well as a number of other kinases including protein kinase C (PKC), tyrosine kinase, mitogen-activated protein kinase (MAPK), and casein kinase 1 (22, 82–84). These protein kinases, through triggering the phosphorylation of particular amino acids on the tail of Cx43, regulate gap junctions (assembly into plaques, stability at the cell surface, gap junction plaque disassembly, and cell–cell communication). Specifically, there are at least 11 serine and 2 tyrosine residues on the C-terminal tail of Cx43 (Cx43-C-terminus) that, when phosphorylated, result in either an increase in gap junction function (S325, S328, S330, S364/365, and S373) or the downregulation of gap junction activity (Y247, S255, S262, Y265, S279/282, and S368) (85–87). These observations have been made mainly from studies in clonal lines of murine fibroblasts (L929 cells), fibroblasts derived from Cx43 knockout and wild-type mice, rat liver epithelial cells (T51B), HeLa cells, and rat primary granulosa cells (88). It should be noted that ACTH-mediated activation of either S364, S365, S369, S373, or other possible amino acids of the C-terminal tail of Cx43 has not, to our knowledge, been examined. Furthermore, protein kinase-mediated phosphorylation of the C-terminus of Cx43 has been demonstrated to play a role in gap junction plaque internalization and annular gap junction vesicle formation (89, 90). Specifically, MAPK, casein kinase 1, and PKC activation increases the number of annular gap junction vesicles while decreasing gap junctions on the cell surface (69, 80, 89–92). In the case of PKC-mediated hyperphosphorylation of Cx43, it is thought that phosphorylation makes this connexin more vulnerable to proteolytic degradation, thus decreasing gap junction-mediated communication. The interplay between the various kinase pathways in Cx43 phosphorylation and cell–cell communication have not been elucidated in the adrenal cortex. Such studies, however, are needed if we are to fully understand the role of ACTH in regulating those endocrine cell responses that are dependent on the communication of molecules between cells. Certainly, in the adrenal gland, there is a need for cells to cooperate and get information from one another. Gap junctions provide this function, and when gap junctions are lost from a population, pathological conditions, including cancers, are thought to develop (42, 93, 94).

### LOSS OF GAP JUNCTIONS AND ADRENAL CORTEX CANCER

The loss of gap junction function has been implicated in tumor development (93–99). Most tumors associated with the adrenal cortex develop in the ZF (100) where, under normal conditions, gap junctions tend to be large and abundant (6, 40, 42). The most common types of adrenal gland tumors are adrenal adenomas, which are non-cancerous tumors of the cortex (101, 102). Adrenocortical carcinoma, cancer of the adrenal cortex, is rarely observed; however, patients with certain inherited genetic disorders are at a higher risk of developing this cancer (102).

In the adrenal cortex, a decrease in gap junction expression has been correlated with the stage of tumor differentiation/progression (42). Specifically, in studies to characterize gap junctions during tumorigenesis, the number of gap junctions observed per cell was quantitated and compared in normal human cells of the adrenal gland ZF, as well as cells from adenoma and carcinoma adrenal gland tissues. Cells from the ZF of normal adrenal glands displayed a much higher number of gap junctions per cell (13.78 ± 1.93 SEM) than when compared with benign adrenocortical adenomas (4.6 ± 1.17 SEM; *p* ≤ 0.05). The number of gap junctions demonstrated between cells of malignant adrenocortical tumors (1.42 ± 0.58 SEM; *p* ≤ 0.05) were significantly lower than both the normal and benign cells (42). These observations are consistent with the hypothesis that a capacity for increase in proliferation coupled with the loss of terminal differentiation as the tumor progresses from non-malignant to malignant reflects the loss of gap junction-mediated communication of growth regulatory molecules between cells. Further, it has been suggested that the increased capacity for tumor metastasis may result from the loss of the cell–cell adhesion that is provided not only by adhesion junctions but also by gap junctions (103–105). Interestingly, exogenous Cx43 can decrease cell proliferation and contribute to reversion of the transformed phenotype (106, 107). Both observations are suggestive of a relationship between gap junctions, differentiation, and proliferation. There is a hope that the induction of gap junctions in malignant cells will provide a novel therapeutic strategy for treating adrenal cancer and, in addition, pharmacological methods and manipulations designed to increase gap junctions may someday serve as a therapeutic method.

While some investigators have suggested the loss of gap junctions in adrenal tumor cell populations, others have demonstrated that metastatic tumors are correlated with an increase in the expression of connexins dependent on the tissue type (108–111). In few cases, tumor cells are capable of communication with other tumor cells through gap junctions; however, they do not communicate with normal cells (112). The difference in the detection of changes in some studies compared with others may reflect the complex nature of cancers in addition to the adrenal zones of origin of the various tumors. Further investigations of the relationship between gap junction expression and tumor progression are needed. This is specifically true when studying the adrenal gland, given the number of adrenal incidentalomas that are commonly detected and the need to differentiate benign from malignant detections. The reported lack of gap junctions in the carcinoma cell population, if found to be widespread in different tumor populations, could be used as an additional method of determining the capacity for metastasis and as an indicator of the rate at which the tumor may proliferate. Furthermore, if cancer cells of the adrenal are capable of communicating with one another but not with normal cells as suggested by Yamasaki in other tissues (112), it would be possible to design protocols that use gap junction-mediated communication to selectively kill the cancer cells while leaving the normal healthy cells intact, the bystander effect (113–115).

## CROSS TALK BETWEEN CELLS OF THE CORTEX AND MEDULLA

It is becoming clear that the cells of the medulla and the cortical cells are physically interwoven with one another (116) and, thus, the possibility of gap junction-mediated communication between chromaffin cells and adrenocortical cells may exist. In humans, as well as other mammals, for example cortical cells can be found within the medulla in contact with chromaffin cells (116–118). Furthermore, inner cortical cells of the ZR are in direct contact with the chromaffin cells of the medulla (1), and gap junctions between these cells could possibly provide a mechanism for direct communication of information. Certainly, there is a functional relationship between the cortical and medullary cells, since the adrenal androgens, particularly dehydroepiandrosterone (DHEA), which is produced in ZR following ACTH stimulation, can inhibit chromaffin cell proliferation and differentiation (119, 120). In addition, cells of the cortex may influence catecholamine release from chromaffin cells while the chromaffin cells may regulate adrenal cortical steroid hormone production (10, 116, 121). Gap junctions may provide a mechanism for some of the functional interdependency, especially, among those cells at the medullary–cortical interface area and among islands of cortical cells located within the medulla.

In the medulla, several different connexin types (Cx29, Cx36, Cx43, and Cx50) have been reported to be expressed, depending on the species (41, 122). While some investigators have not reported gap junction plaques in the medulla, others have demonstrated clusters of medullary cells that express Cx43 antigens (5). It is thought that gap junctions in the medulla play a role in the catecholamine (epinephrine and norepinephrine) secretory process and, thus, helps in mediating the body's response to stress (54, 123–125). In addition, stress causes a well-orchestrated, cascade of events that result in the release of a number of other hormones, including glucocorticoids from the adrenal cortex (126). Gap-junctional coupling may complement electrical coupling to enhance communication of signals needed for synchronized cellular hormone release in response to ACTH or low synaptic activity during stress (125).

### SUMMARY

Gap junctions are thought to be important in regulating cell population growth, cell morphology, differentiation, cell migration, wound healing, and cell function (2, 93, 108). By analyzing the frequency, distribution, and function of these junctions within intact adrenal glands as well as in adrenal cells in culture following ACTH stimulation, the role of connexins in these processes may be clarified. The specific spatial abundance of gap junction proteins in the adrenal cortical zones is intriguing with regard to its functional implications. It can be suggested that cells of the zona glomerulosa, which express fewer gap junctions than the two other cortical zones, may be less dependent on cell–cell communication for normal function than the cells of the ZF or ZR. Further, a relationship between proliferation rates, steroidogenesis, and gap junction presence or absence in the gland has been suggested (**Figure 7)**. Studies to demonstrate the effects of gap junction overexpression and inhibition in the adrenal glands are needed, if we are to resolve some of the questions of how gap junctions function as adrenal cells undergo the morphological and proliferative changes associated with the establishment and maintenance of cortical zonation and hormone-mediated steroidogenesis.

Intact adrenal glands and adrenal cell cultures provide us with a valuable paradigm for studying gap junctions and the cellular mechanisms involved in hormone responses. Indeed, study of adrenocortical cells *in vivo* and *in vitro* is important not only for an understanding of adrenal gland function *per se* but also for providing insight into the foundation of cell communication and biological response in other tissues. But, beyond cell communication, it is becoming apparent that gap junctions play a role in providing adhesive forces needed to hold cells together (103–105).

In addition to gap junction-mediated communication and adhesion, the gap junction internalization process that results in increased annular gap junction vesicles within the cytoplasm and fewer surface gap junction plaques could be important to events in the adrenal gland. Annular gap junctions were reported in the adrenal gland as early as 1988 with transmission electron microscopy (49). It has been demonstrated that annular gap junctions undergo degradation (13, 51, 52). Relatively large numbers of annular gap junctions occur in the adrenal gland, and it could be postulated that gap junction turnover is high in this tissue. Rapid turnover could be needed for migration of the cells to maintain the cortical zones during cytogenesis, for example. Such suggestions are not without precedent. In the ovary, for example, gap junctions are thought to allow the movement of molecules to the egg from the follicular granulosa cells (60, 61). In the case of the ovary, phosphorylation of the C-terminal tail of connexin has been demonstrated to be regulated by the peptide hormone, luteinizing hormone (127). Large numbers of annular gap junctions are reported during ovulation, and it is thought that if gap junctions are not internalized from the surface of the granulosa cells that the egg will remain "trapped" within follicle and ovulation will not occur (128). Gap junctions in a similar way could potentially retard the migration of cells from one area to another (centripetal migration) by tethering the cells to one another. Inhibiting cell migration through the granulosa cell layer of the follicle, in the case of the ovary, or as cells migrate in the adrenal gland could keep cells connected to one another if gap junctions fail to internalize. Such possibilities, however, are yet to be demonstrated.

### REMAINING QUESTIONS

It is clear from early studies that there is a relationship between gap junctions and ACTH-mediated responses in the adrenal cortex. There are, however, very few recent articles available

on gap junctions in the adrenal cortex. Yet, there are important questions that remain to be answered. For example, in human conditions with diminished circulating ACTH levels, are there changes in the expression or distribution of adrenal cortical gap junctions, as seen following hypophysectomy in rodents (30)? There is dramatic remodeling and relocation of gap junction plaques in the diabetic heart (129). Would the remodeling of gap junctions in the adrenal occur in this and in other endocrine related diseases? Adrenal cortical steroidogenesis is influenced mainly by the activation of PKA (59, 64). In tissues, other than the adrenal, PKA has been shown to phosphorylate the C-terminal tail of Cx43 gap junction proteins (22, 78–81). Such PKAmediated connexin phosphorylations have been demonstrated to be critical in regulating gap junction assembly and stability (22, 78–81). In the adrenal cortex following ACTH treatment, it is presumed that gap junction protein phosphorylation occurs. However, which Cx43 C-terminal tail amino acids are phosphorylated is unknown. Further, the interplay between PKA and the other protein kinases (PKC, tyrosine kinase, MAPK, and casein kinase 1) in the regulation of gap junctions and adrenal cortical functions needs to be investigated. It has become clear that connexins can have multiple regulatory functions, which depend upon their phosphorylated state (22, 82, 84). Unfortunately, the role of protein kinase-mediated connexin phosphorylation in the adrenal cortex, although described in other tissues, has not been investigated, to our knowledge, in the adrenal cortex. However, one would predict that as cells alter their function that the need for gap junction-mediated cell communication, and thus gap junction expression and phosphorylation, may also change. Moreover, the molecules moving between cells may change. One molecule that has been demonstrated to transfer between gap junctions of adrenal cells to regulate steroidogenesis is cAMP (59, 68). But surely, more than this, one molecule is moving between the cells of the adrenal. The abundance of gap junctions in the inner cortex would suggest a high demand for communication of molecules in these zones. The following question has yet to be answered: how movement of molecules between cells serves to regulate the cascade of events involved in proliferation but more importantly in differentiation of the cells such that transcription factors needed for the production of cortisol in one zone of the adrenal but for androgens in another zone.

The study of adrenocortical cells *in vivo* and *in vitro* is important not only for an understanding of adrenal gland function *per se* but also for providing insight into the foundation of cell communication and biological response in other tissues. Further study will

### REFERENCES

elucidate how gap junctions and cell–cell communication as well as gap junction-mediated adhesion is related to adrenal gland development cell differentiation and steroidogenic function.

### AUTHOR CONTRIBUTIONS

CB wrote sections of the manuscript, prepared figures, and edited the manuscript. SM wrote sections of the manuscript, prepared figures, and edited the manuscript.

### ACKNOWLEDGMENTS

This review provides only a sampling of the literature that is available on this topic, and we apologize to authors whose original works have not been cited. Work in the authors' laboratory is supported by funds from the National Science Foundation (NSF, grant# MCB-0444398) to SM.

elevated mitogen-activated protein kinase (p44/Erk1, p42/Erk2) activation. *Carcinogenesis* (2004) 25:669–80. doi:10.1093/carcin/bgh071

interactions. *J Clin Endocrinol Metab* (1994) 78:225–32. doi:10.1210/ jcem.78.1.7507122

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Bell and Murray. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Adrenal Gland Microenvironment and Its Involvement in the Regulation of Stress-Induced Hormone Secretion during Sepsis

#### *Waldemar Kanczkowski1 \*, Mariko Sue1 and Stefan R. Bornstein1,2*

*1Department of Internal Medicine III, Technische Universität Dresden, Dresden, Germany, 2Department of Endocrinology and Diabetes, King's College London, London, UK*

Survival of all living organisms depends on maintenance of a steady state of homeostasis, which process relies on its ability to react and adapt to various physical and emotional threats. The defense against stress is executed by the hypothalamic–pituitary–adrenal axis and the sympathetic–adrenal medullary system. Adrenal gland is a major effector organ of stress system. During stress, adrenal gland rapidly responds with increased secretion of glucocorticoids (GCs) and catecholamines into circulation, which hormones, in turn, affect metabolism, to provide acutely energy, vasculature to increase blood pressure, and the immune system to prevent it from extensive activation. Sepsis resulting from microbial infections is a sustained and extreme example of stress situation. In many critical ill patients, levels of both corticotropin-releasing hormone and adrenocorticotropin, the two major regulators of adrenal hormone production, are suppressed. Levels of GCs, however, remain normal or are elevated in these patients, suggesting a shift from central to local intra-adrenal regulation of adrenal stress response. Among many mechanisms potentially involved in this process, reduced GC metabolism and activation of intra-adrenal cellular systems composed of adrenocortical and adrenomedullary cells, endothelial cells, and resident and recruited immune cells play a key role. Hence, dysregulated function of any of these cells and cellular compartments can ultimately affect adrenal stress response. The purpose of this mini review is to highlight recent insights into our understanding of the adrenal gland microenvironment and its role in coordination of stress-induced hormone secretion.

Keywords: stress system, hypothalamic–pituitary–adrenal axis, immune–adrenal crosstalk, ACTH, glucocorticoids

### INTRODUCTION

In order to survive all, living organisms must maintain a steady state of homeostasis. This unique capacity to react and adapt to various physical and emotional threats in higher vertebrates is mediated by a coordinated action of the nervous, endocrine, and immune systems, known as stress system (1). Activation of the stress system, which is composed of the hypothalamic–pituitary–adrenal (HPA) axis and the sympathetic–adrenal medullary system, leads to increased synthesis and release of glucocorticoids (GCs) and catecholamines (CAs) from the adrenal cortex and medulla, respectively (2–4). The main function of these hormones is maintenance and restoration of basal and stress-related

#### *Edited by:*

*Nicole Gallo-Payet, Université de Sherbrooke, Canada*

#### *Reviewed by:*

*Tamas Kozicz, Radboud University Nijmegen, Netherlands Olivier Lesur, Université de Sherbrooke, Canada*

#### *\*Correspondence:*

*Waldemar Kanczkowski waldemar.kanczkowski@ uniklinikum-dresden.de*

#### *Specialty section:*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology*

*Received: 16 September 2016 Accepted: 29 November 2016 Published: 14 December 2016*

#### *Citation:*

*Kanczkowski W, Sue M and Bornstein SR (2016) Adrenal Gland Microenvironment and Its Involvement in the Regulation of Stress-Induced Hormone Secretion during Sepsis. Front. Endocrinol. 7:156. doi: 10.3389/fendo.2016.00156*

body homeostasis. This protective action of adrenal hormones is mostly accomplished by their metabolic, cardioprotective, and anti-inflammatory actions. In particular, both GCs and CAs are known to acutely enhance plasma glucose levels and promote an increased cardiac output and high blood pressure, while protecting against excessive inflammation (5–8).

Secretion of adrenal hormones during stress-free conditions is regulated centrally and is characterized by ultradian rhythms (9). An early morning release of adrenocorticotropic hormone (ACTH) into circulation is enhanced by increased concentrations of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) in the hypophyseal portal system of anterior pituitary (10, 11). Consequently, elevated plasma levels of ACTH stimulate adrenocortical cells to produce and release GC hormones. In turn, as a part of negative feedback, GCs act directly on the pituitary gland to reduce ACTH secretion and on hypothalamic neurons to reduce CRH release (11, 12). Systemic rise in CA levels is in turn initiated by activation of the sympathetic nervous system and splenic nerves innervating the adrenal medulla (13, 14).

One of the extreme examples of sustained and severe physical stress is sepsis syndrome. It is characterized by abnormal host response to infection, resulting in systemic inflammation that frequently culminates in a life-threatening dysfunction of multiple organs (15, 16). Severe sepsis remains the leading cause of mortality worldwide, and its incidence is increasing (17, 18). An intact function of the stress system, and in particular the activation of GC and CA production, is critical to survive this adverse condition (19, 20). During sepsis, this homeostatic function of the stress system is frequently impaired with mechanisms remaining largely unknown (21, 22). Consequently, many critically ill patients demonstrate suppressed ACTH and CRH levels while having normal or elevated cortisol levels (22). Hence, a key involvement of pituitary-independent factors was proposed (2). Among these, reduced GC metabolism and activation of local adrenal microenvironment seem to play crucial role (23, 24) (**Figure 1**).

As regulation of the stress system and importance of impaired metabolism of GCs during sepsis were recently presented and discussed in many review articles (25–27), the main purpose of this mini review is to highlight recent insights into our understanding of the role local adrenal gland microenvironment in the regulation of stress-induced hormone secretion during sepsis.

### ADRENAL GLAND MICROENVIRONMENT

Within adrenal gland, two embryonically different tissues coexist: mesodermally derived, steroid-producing cortex and ectodermally derived, CA-producing medulla. Within these two environmental niches, interplay between various cells takes place including adrenocortical and chromaffin cells, neuronal cells, immune cells, endothelial cells, and glia cells (28). The adrenal gland is a source of many bioactive substances including steroid hormones, CAs, cytokines, neurotransmitters, and neuropeptides (**Table 1**). These substances are known to interact with various cell types within adrenal gland itself, thereby influencing its function during stress conditions and disease (24, 28).

## Adrenal Cortex and Medulla Interactions during Sepsis

Ample evidence exists suggesting a bidirectional interaction between adrenal cortex and the medulla (28). Despite a classical view on the adrenal gland anatomy, demonstrating a clear separation of the cortex and medulla, it has been well documented that these two endocrine tissues are morphologically intermingled (29, 30). This close localization, in turn, enables direct cell–cell and paracrine interactions between adrenocortical and chromaffin cells involving their secretory products.

Consequently, the adrenal cortex-derived GCs were found to enhance synthesis of CAs both *in vitro* (31) and *in vivo* (32–34). GCs were found to execute these effects by upregulating expression of phenylethanolamine *N*-methyltransferase (PNMT), which gene encodes for an enzyme that catalyzes the synthesis of epinephrine from norepinephrine. Furthermore, studies using various knockout mice, which were deficient in steroidogenic factor-1, glucocorticoid receptor, or corticotropin-releasing hormone type 1 receptor, clearly demonstrated that lack of one of the these key steroidogenic regulators impair PNMT gene expression and thus basal and stress-induced epinephrine production (35–38). Similarly, an intact function of adrenal medulla is important to maintain adrenocortical function. It has been shown that coculture of bovine adrenocortical cells with chromaffin cells enhanced a basal GC secretion by 10-fold (39). Furthermore, CAs were found to enhance production of various steroids including cortisol, aldosterone, and androstenedione, through mechanism involving upregulation of steroidogenic acute regulatory protein (28, 40, 41).

During sepsis, an enhanced production of epinephrine and norepinephrine (42–44) and increased secretory capacity of adrenal chromaffin cells were reported (45). Unlike expression of tyrosine hydroxylase, which enzyme is involved in first step of CA biosynthesis, upregulation of PNMT gene expression in adrenal medulla was found to be regulated independently of splanchnic nerve stimulation (28). Instead, an extremely high concentration of GCs was reported to be involved in regulation of PNMT expression during sepsis (46). Consequently, stress-induced expression of PNMT is strongly attenuated in CRH-deficient mice (47).

In addition, adrenal chromaffin cells release many active neuropeptides and transmitters, including neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), or substance P, with known stimulatory action on GCs production (48, 49). For example, VIP, which levels increase during experimental endotoxemia in adrenal medulla, was found to stimulate DHEA, testosterone, androstenedione, cortisol, and aldosterone secretion in adrenocortical cells (48–52). Among other mediators found in adrenal medulla, which may influence GC production during sepsis, are angiotensin, AVP, CRH, ACTH, and apelin (53–55). Although direct stimulatory effects of angiotensin 2, CRH, and ACTH on adrenal hormone production are well described, the degree to which each of these peptides contributes to hormone production during sepsis conditions has not been fully elucidated yet (2, 56). Another interesting peptide found recently to be expressed in the adrenal medulla is apelin. Apelin is a neurohormone, which acts through its receptor APJ expressed in various organs including

#### TABLE 1 | Intra-adrenal interaction involved in a regulation of adrenal stress response.

hypothalamic neurons, anterior pituitary, and adrenal gland. It was recently found to modulate neuroendocrine response to stress through stimulation of secretion of both ACTH and corticosterone directly and *via* AVP- and CRH-dependent pathways (54, 55, 57).

Recently, a population of nestin-positive glia-like multipotent stem cells was identified in adrenal medulla of nestin-GFP transgenic mice (58). Interestingly, when subjected to chronic stress, this population was able to give rise to new chromaffin cells, suggesting a direct involvement of these cells in the adrenal stress adaptation (58–60). It is therefore very interesting to investigate the fate of this cells and their potential interaction with adrenocortical cells during sepsis conditions.

In summary, an intact function of both adrenal cortex and adrenal medulla is of pivotal importance in the regulation of adrenal stress response. Consequently, any disorders or medications that can attenuate adrenal cortex function will also ultimately affect CAs production by chromaffin cells and *vice versa* potentially impacting the outcome of many stress-related disorders (19).

### Immune–Adrenal Crosstalk

In the last 40 years, it has become evident that immune and endocrine systems interact with each other at the level of adrenal gland and that this interaction is crucially involved in the regulation of adrenal gland function during normal and stress conditions (61). This immune–endocrine crosstalk is mostly executed by bidirectional action of paracrine factors, such as steroid hormones, CAs, and various vasoactive or proteolytic enzymes, as well as by direct cell–cell contacts and activation of toll-like receptors (TLRs) (28, 62).

### Immune–Adrenal Interaction at the Level of Adrenal Cortex

During non-stress conditions, many immune cells can be found in the innermost zone of adrenal cortex known as *zona reticularis*; these are particularly tissue macrophages (63), dendritic cells (64), mast cells (65), and lymphocytes (66). However, some of them, especially macrophages and mast cells, were also reported in other parts of the adrenal gland including subcapsular region and in the adrenal medulla (64). In non-stress conditions, adrenal-resident immune cells play important homeostatic functions by sensing pathogens, removing apoptotic cells, and promoting tissue remodeling through, e.g., secretion of growth factors (24, 67). Recently, a new population of adrenal macrophages was identified based on the high expression of a complement receptor immunoglobulin, CRIg, which molecule is known to mediate phagocytosis of pathogens and apoptotic cells (68). During systemic inflammation induced either by LPS injection or CLP, highly dynamic changes within adrenal immune cell populations can be observed. For instance, it has been shown that already within first 3 h after administration of LPS or induction of peritonitis in rodents, a rapid infiltration of neutrophils takes place, while the number of local dendritic cells and macrophages declines (69–71).

Using electron microscopy, close cell–cell localization between local immune cells and surrounding adrenocortical, chromaffin, or endothelial cells was demonstrated (64, 66). This in turn enables various bidirectional and direct interactions between each cell type. For example, a functional crosstalk between adrenocortical cells, expressing major histocompatibility complex class II molecules (MHC/HLA), and leukocytes was described. Indeed, a co-incubation of T cells with primary cultures of human adrenocortical cells promoted adrenal androgen and cortisol secretion (66). Besides direct interaction, various immune cells were found to regulate adrenal hormone production in a paracrine way through secretion of cytokines, such as interleukins (IL) 1 and 6 (72), and pro-opiomelanocortin (POMC)-derived peptides, e.g., ACTH and biogenic amines (28, 73).

The ample experimental evidence exists demonstrating that cytokines are critically involved in ACTH-independent activation of GC production during sepsis (72). For example, injection of IL-1β enhanced GC production in hypophysectomized rats (74). Furthermore, an impaired synthesis of GCs after LPS injection was observed in mice receiving sera against tumor necrosis factor (TNF)-α and IL-6 and, to a lesser extent, IL-1β- or in IL-6-deficient mice (75, 76). However, it has been demonstrated that cytokine levels found normally in plasma are far too low to induce hormone production from adrenocortical cells. Therefore, the main source of cytokines must come from the adrenal gland itself (28). Sustained production of cytokines in highly antiinflammatory environment of the adrenal gland during systemic inflammation was found to be enabled by increased expression of migration inhibitory factor (77). During sepsis, various cell types present within adrenal gland microenvironment may be a potential source of pro-inflammatory cytokines. For example, adrenocortical cells were found to express several TLRs and secrete several cytokines in response to bacterial ligands (78, 79). However, in a recent study, inactivation of myeloid but not adrenocortical TLR signaling resulted in significant reduction of intra-adrenal cytokine levels and activation of the HPA axis after LPS administration (80). The latter observation suggests that immune cells are the key sources of cytokines in the adrenal gland during sepsis. Due to high expression of both cytokine and GC receptors in adrenocortical and immune cells, respectively, cytokines and adrenal hormones are known to regulate production in each other. For example, IL-1β or IL-6 was found to increase adrenal hormone production, whereas TNF stimulation demonstrated rather opposite effect (81–83). In turn, adrenal steroid hormones are known to influence immune cells function. In particular, it has been demonstrated that in naïve immune cells, GCs can activate several inflammatory-related genes, e.g., TLRs, yet in cells treated with LPS, they inhibit inflammation (6). In a recent study, a chronic exposure to GCs was found to cause a shift in the innate–adaptive balance of the immune response, particularly, influencing the chemokine–chemokine receptor networks (84). Besides cytokines, other biologically active substances, such as serotonin (5-HT) or histamine, which are stored by mast cells show stimulatory effects on adrenal steroidogenesis (85, 86).

During prolonged sepsis, the immune–adrenal interaction may also result in the suppression of adrenal hormone production. Despite their important role in host defenses against bacterial infections limiting bacterial spread in circulation (87), a prolonged exposure of adrenal cells to neutrophil-derived antimicrobial agents, such as ROS and proteolytic enzymes secreted during formation of neutrophil extracellular traps, results in tissue damage (88). In addition, neutrophils were found to secrete corticostatins, e.g., α-defensins, which substances are known to interfere with ACTH-mediated increase in adrenocortical hormone production (89). In a recent study using developmental endothelial locus 1 (Del-1)-deficient mice (90), an association between higher amount of infiltrating neutrophils and impaired adrenal corticosterone production after systemic LPS administration was found (91).

### Immune–Adrenal Interaction at the Level of Adrenal Medulla

Activation of the sympathetic nerve system during sepsis results in enhanced production and secretion of CAs from the adrenal chromaffin cells (44) through a mechanism involving an increased release of Ca2+ from endothelial reticulum (45). An early increase in endogenous CA level plays an important protective role as its absence due to either pharmacological or surgical intervention resulted in induction of hypotension and increased mortality of rats during experimental endotoxemia (20, 43). One of the mechanisms involved in the protective role of CAs, besides wellknown vasopressor function, is the anti-inflammatory action on various immune cells. The immunomodulatory effects of CAs are mediated *via* their direct interactions with β-adrenergic receptors (ARs) expressed by a variety of immune cells. In particular, it has been shown that CAs promote IL-10 secretion while decreasing production of pro-inflammatory TNF cytokine in LPS-treated macrophages (92). Furthermore, incubation of neutrophils with CAs decreased NO production and ROS generation (93). However, these immune-suppressive effects of CAs should be carefully interpreted as they may correspond predominantly to the leukocyte populations or be restricted to β-AR activation. In fact, recently both epinephrine and norepinephrine were shown to increase IL-6 production in endothelial cell line: HMEC-1 and in human skin microvascular endothelial cells (94). It has been also found that activation of α-ARs in macrophages by CA may potentiate inflammation by increasing TNF-α production (95). Recently, phagocytes were shown to be able not only to secrete CAs in response to LPS stimulation but also to possess all major CA-generating and -degrading enzymes required for their production and inactivation (96). This discovery adds additional complexity to already complex immune–chromaffin cell interaction. During sepsis, the adrenal medulla shows high degree of immune infiltration (69). This suggests that overactivation of immune cells could potentially impair production and secretion of adrenomedullary hormones or induce structural damage. Indeed, during experimental sepsis induced by CLP, systemic inflammation resulted in a strong apoptosis in the adrenal chromaffin cells, which process required activation of receptors for complement C5a (97). The latter observation suggests that during progression of sepsis, an increased activation of immune system promoting apoptosis of chromaffin cells may impact stress-induced CA production.

### Adrenal Vasculature

Vascular system plays a key role in the proper functioning of many vital organs during sepsis. Besides enabling the secretion of steroid hormones and CAs into circulation, adrenal vasculature was found to control leukocyte infiltration through expression of adhesion molecules and Del-1 protein (91).

Adrenal cortex is characterized by high density of endothelial cells that stay in close vicinity with steroid-producing cells. Consequently, both adrenal hormones and endothelial cellderived products were shown to influence function of adrenal vasculature and steroid-producing cells, respectively. For example, human adrenocortical cells increase production of aldosterone and cortisol once incubated with endothelial cell-conditioned media. Although the exact composition of the latter media was not determined, this stimulatory effect was found to require cAMP, but not PKA, pathway (98). Endothelial cells are known to produce several factors that, e.g., nitric oxide (NO) or endothelin-1, could potentially influence adrenal hormone production. In particular, in rat and human adrenocortical cells, it has been found that endothelin-1 can directly promote GC synthesis and potentiate angiotensin II and ACTH-induced aldosterone production (99). Besides endothelin-1, nitric oxide (NO) was found to exert stimulatory effect on adrenal steroidogenesis by mediating the acute response to ACTH (28). In fact, blocking of NO production either by using NO synthase inhibitor or NO scavengers resulted in decreased ACTH-mediated corticosterone release (100). However, in other studies using perfused adrenal glands administration of l-arginine, the substrate for nitric oxide synthesis did not change corticosterone response to ACTH, suggesting an indirect modulatory action of NO (101).

During sepsis, in many critically ill patients, an increased size of adrenal gland resulting from the hypervascularization and increased blood flow and adrenal hyperplasia were found to positively correlate with survival rate (102). Although an intact activity of splanchnic nerve is crucial in regulation of adrenal blood flow, other factors such as VIP, NPY, or neurotensin were found also to be involved (103, 104). Furthermore, a positive correlation between an increased blood flow and enhanced adrenal steroidogenesis was demonstrated (105). Indeed, an enhanced adrenal blood flow was found to participate in angiotensin II- and ACTH-induced aldosterone and cortisol production. This stimulatory action on

### REFERENCES

adrenal vascular response to angiotensin II and ACTH was found to be mediated by vasoactive substances released from granules of mast cells (106). However, more recently, additional involvement of the cytochrome 450 in controlling the adrenal blood flow after AII and ACTH was demonstrated in rats (24, 107).

### SUMMARY

Sepsis and septic shock strongly impacts body homeostasis, which if not sufficiently counteracted by activated stress system results in increased mortality of critically ill patients (56, 108, 109). During chronic phase of sepsis, elevated adrenal hormone secretion was reported to be mediated by pituitary-independent mechanisms including activation of intra-adrenal microenvironment (22, 23). An increasing amount of experimental studies support the key involvement of several cell types and cellular systems present within adrenal gland microenvironment in the sustained production of adrenal hormones during sepsis. Consequently, any impairment in function of these systems can ultimately affect adrenal stress response (2, 24).

### AUTHOR CONTRIBUTIONS

WK, MS, and SB have all made substantial, direct, and intellectual contribution to the work and approved it for publication.

### FUNDING

This work was supported by grants of the Deutsche Forschungs Gemeinschaft (DFG) to WK (KA 3013/2-2) and SB (BO 1141/11-2).

19. Chan CM, Mitchell AL, Shorr AF. Etomidate is associated with mortality and adrenal insufficiency in sepsis: a meta-analysis. *Crit Care Med* (2012) 40:2945–53. doi:10.1097/CCM.0b013e31825fec26

20. McKechnie K, Dean HG, Furman BL, Parratt JR. Plasma catecholamines during endotoxin infusion in conscious unrestrained rats: effects of adrenal demedullation and/or guanethidine treatment. *Circ Shock* (1985) 17:85–94.

by coculture with chromaffin cells. *Endocrinology* (1998) 139:772–80. doi:10.1210/en.139.2.772

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer OL and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

*Copyright © 2016 Kanczkowski, Sue and Bornstein. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Role of ACTH in the Interactive/ Paracrine Regulation of Adrenal Steroid Secretion in Physiological and Pathophysiological Conditions

*Hervé Lefebvre1,2,3\*, Michaël Thomas1,2, Céline Duparc1,2, Jérôme Bertherat4,5 and Estelle Louiset1,2*

*1U982, Laboratory of Neuronal and Neuroendocrine Differentiation and Communication, INSERM, Institute for Research and Innovation in Biomedicine, Mont-Saint-Aignan, France, 2Normandie Université, UNIROUEN, Rouen, France, 3Department of Endocrinology, Diabetes and Metabolic Diseases, University Hospital of Rouen, Rouen, France, 4U1016, INSERM, Institut Cochin, Paris, France, 5Department of Endocrinology and Metabolic Diseases, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France*

#### *Edited by:*

*Nicole Gallo-Payet, Université de Sherbrooke, Canada*

#### *Reviewed by:*

*Francisco Gaytán, University of Córdoba, Spain Andre Lacroix, Université de Montréal, Canada Duarte L. Pignatelli, Institute of Molecular Pathology and Immunology of the University of Porto, Portugal*

> *\*Correspondence: Hervé Lefebvre<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology*

*Received: 30 March 2016 Accepted: 07 July 2016 Published: 20 July 2016*

#### *Citation:*

*Lefebvre H, Thomas M, Duparc C, Bertherat J and Louiset E (2016) Role of ACTH in the Interactive/Paracrine Regulation of Adrenal Steroid Secretion in Physiological and Pathophysiological Conditions. Front. Endocrinol. 7:98. doi: 10.3389/fendo.2016.00098*

In the normal human adrenal gland, steroid secretion is regulated by a complex network of autocrine/paracrine interactions involving bioactive signals released by endothelial cells, nerve terminals, chromaffin cells, immunocompetent cells, and adrenocortical cells themselves. ACTH can be locally produced by medullary chromaffin cells and is, therefore, a major mediator of the corticomedullary functional interplay. Plasma ACTH also triggers the release of angiogenic and vasoactive agents from adrenocortical cells and adrenal mast cells and, thus, indirectly regulates steroid production through modulation of the adrenal blood flow. Adrenocortical neoplasms associated with steroid hypersecretion exhibit molecular and cellular defects that tend to reinforce the influence of paracrine regulatory loops on corticosteroidogenesis. Especially, ACTH has been found to be abnormally synthesized in bilateral macronodular adrenal hyperplasia responsible for hypercortisolism. In these tissues, ACTH is detected in a subpopulation of adrenocortical cells that express gonadal markers. This observation suggests that ectopic production of ACTH may result from impaired embryogenesis leading to abnormal maturation of the adrenogonadal primordium. Globally, the current literature indicates that ACTH is a major player in the autocrine/paracrine processes occurring in the adrenal gland in both physiological and pathological conditions.

Keywords: ACTH, aldosterone, cortisol, Cushing's syndrome, aldosterone-producing adenoma, hyperplasia, adrenocortical cells, VEGF

### INTRODUCTION

The adrenal cortex is a heterogeneous tissue that not only contains steroidogenic cells but also hosts various cell types that are able to locally release a wide variety of bioactive signals. This histological organization results in a complex interactive network that participates in the regulation of both basal and ACTH-induced corticosteroidogenesis. The intracortical sources of regulatory factors include chromaffin cells arranged in cords or islets, nerve fibers originating from extraadrenal neurones or cell bodies located in the adrenal medulla, cells of the immune system, including lymphocytes, Lefebvre et al. ACTH in Adrenal Glands

macrophages/monocytes and mast cells, endothelial cells, and adipocytes. These autocrine/paracrine mechanisms have been extensively reviewed during the past years (1–5). However, their physiological role, especially their exact contribution to the regulation of corticosteroid synthesis remains a matter of debate although some data indicate that intraadrenal regulatory signals may mediate parts of the biological effects of ACTH on the adrenal cortex. Interestingly, adrenocortical neoplasms associated with steroid hypersecretion exhibit molecular and cellular defects that tend to reinforce the potency of paracrine factors to activate corticosteroidogenesis. For instance, abnormal expression of ACTH has been reported in adrenocortical cells in both tumors and hyperplasias responsible for hypercortisolism (6–11).

In the present review article, we have updated the current knowledge on the role of ACTH in the cell-to-cell communication processes occurring in the adrenal cortex in both physiological and pathological conditions. We will also discuss the potential interest of the intraadrenal ACTH regulatory loop for the clinical management of patients with primary adrenal excess of corticosteroids.

### EFFECT OF ACTH ON THE ADRENAL VASCULATURE

The adrenal cortex is a richly vascularized organ. This extensive vasculature is essential for delivery of tropic hormone and steroid hormones precursors to the gland and secretion of mature hormones into the blood flow. Furthermore, the establishment of such dense vascular network ensures that every adrenocortical cell is in contact with at least one endothelial cell (12, 13). This remarkable histological organization allows paracrine regulation of adrenocortical cells by endothelial cells through release of endothelins, adrenomedullin, nitric oxide, and prostacyclin (14). For instance, Rossi et al. have shown that endothelin-1 released by endothelial cells is an important regulator of aldosterone secretion, and may then indirectly influence arterial blood pressure (15, 16). The release of endothelin by adrenocortical sinusoids is thought to mediate the modulation of adrenal steroidogenesis by the adrenal blood (17). Interestingly, ACTH appears able to both act on development and maintenance of the adrenal vasculature and regulate the adrenal blood flow (18, 19), influencing thus steroid production through an indirect effect in addition to its intrinsic steroidogenic action on adrenocortical cells (**Figure 1**).

The modulation of adrenal angiogenesis by ACTH may involve several bioactive signals. Thrombospondins (TSPs) represent a wide family of extracellular proteins consisting of five members, TSP1–5, which can bind multiple cell surface molecules, including heparin sulfate proteoglycans, low-density lipoprotein receptor-related protein, integrins, CD36, and CD47 (20). Owing to the great diversity of their binding partners, TSPs are involved in various biological processes, such as cell adhesion, spreading and migration, and angiogenesis (20, 21). In this respect, TSPs are known to inhibit angiogenesis by preventing migration of capillary endothelial cells. Interestingly, adrenocortical cells release high amounts of TSP2 in response to ACTH (22), suggesting that TSP2 may mediate some of the biological actions of

FIGURE 1 | Direct and indirect effects of ACTH on steroidogenesis in the adrenal gland. ACTH exerts a direct stimulatory effect on adrenocortical cells *via* melanocortin type 2 receptors (MC2R). Activation of MC2R increases secretion of steroid hormones, vascular endothelial growth factor (VEGF), and epoxyeicosatrienoic acids (EETs). Angiogenesis induced by VEGF, an endothelial cell-specific mitogen factor, allows adrenal tissue growth in response to ACTH. Increase in adrenal blood flow elicited by EETs, which are potent vasorelaxant agents, favors steroid production. In rats, ACTH also activates adrenal mast cells inducing local release of histamine and serotonin (5-HT). These factors modulate the tonicity of adrenal arterioles and, thus, the adrenal blood flow that indirectly influences the steroidogenesis. In addition, 5-HT stimulates steroid secretion through activation of different 5-HT receptor subtypes expressed by normal and tumor human adrenocortical cells.

ACTH in the adrenal cortex, especially centripetal adrenocortical cell migration, which is a fundamental process in the dynamic organization and remodeling of the adrenal cortex (23). However, the physiological role of TSP2, which is primarily expressed in zona glomerulosa and zona fasciculata, remains unclear since TSP2-null mice exhibit no alteration in corticosteroid secretion or adrenal development (24). In addition, syndromes of ACTH excess, including Cushing's disease and 21-hydroxylase deficiency, are associated with adrenocortical hyperplasia, a process that underlies active angiogenesis. Conversely, several observations indicate that vascular endothelial growth factor (VEGF) plays a pivotal role in the trophic effects of ACTH on the adrenal vasculature (25). VEGF is a widely expressed cytokine that acts as an endothelial cell-specific mitogen and angiogenic factor. In the bovine adrenal gland, VEGF is expressed in zona glomerulosa and zona fasciculata cells and its release is stimulated by ACTH (26). Upregulation of VEGF by ACTH has also been reported in human adrenal (27). The effect of ACTH on adrenal VEGF production involves transcription-independent mechanisms, including stabilization of VEGF mRNA by the HuR protein (28). Conversely, ACTH suppression by dexamethasone in mice results in progressive decrease of VEGF expression in adrenocortical cells and regression of the vascular network (29). Interestingly, ACTH also stimulates VEGF expression in human fetal adrenocortical cells, suggesting that VEGF is an important mediator of the trophic action of ACTH during the adrenal development (30). Consistently, an increase in VEGF expression in adrenocortical cells has been noticed in the regenerating adrenal cortex in rats (31, 32). It is noteworthy that, in parallel to its effect on adrenal vasculature development *via* local synthesis of VEGF, ACTH also favors adrenal tissue growth through an antiapoptotic action on adrenocortical cells (29) and stimulation of synthesis of growth factors (30). Finally, VEGF may be involved in the capacity of ACTH to induce endothelial fenestration, a phenomenon that favors cell-to-cell interactions (26, 33).

The mechanism by which ACTH modulates the adrenal blood flow is obviously not univocal. Adrenocortical cells themselves are able to release vasorelaxant agents in response to ACTH. These compounds include metabolites of arachidonic acid, such as epoxyeicosatrienoic acids (EETs) (34). In addition, in the rat adrenal gland, capsular mast cells modulate the tonicity of adrenal arterioles and, thus, the adrenal blood flow through local release of histamine and serotonin (5-hydroxytryptamine; 5-HT) (17, 35). Interestingly, rat adrenal mast cells are sensitive to the action of ACTH, suggesting that they may represent an important intermediate in the effect of corticotropin on the adrenal blood flow (35). In humans, the presence of mast cells has been reported in both the subcapsular region of the normal adrenal gland (36) and various types of adrenocortical tumors, including deoxycorticosterone-secreting tumors, aldosterone-producing adenomas (APAs), and adrenocortical carcinomas (37–39). However, there is currently no data reported in the literature indicating that human adrenal mast cells are target cells for ACTH.

### INDIRECT EFFECTS OF ACTH ON CORTICOSTEROIDOGENESIS THROUGH ADRENOCORTICAL CELL SECRETORY PRODUCTS

Adrenocortical cells are important sources of bioactive compounds that are able to modulate steroidogenesis through autocrine/ paracrine actions. In man, it is doubtful that corticosteroid may affect their own secretion in physiological conditions. Conversely, the adrenal cortex is known to express the diverse components of the renin–angiotensin system, leading to local synthesis of angiotensin II and potential autocrine/paracrine stimulation of aldosterone secretion (40). Interestingly, renin production, which primarily occurs in zona glomerulosa cells, is stimulated by ACTH and reduced by hypophysectomy or dexamethasone administration (41, 42). This observation may explain why plasma renin levels are usually not suppressed in patients with overt ACTH-dependent hypercortisolism (43, 44). In fact, it is conceivable that, in this condition, ACTH-induced adrenal renin secretion may compensate inhibition of renal renin synthesis by hypervolemia and hypertension secondary to cortisol excess.

Adrenocortical cells also synthesize and release various cytokines, such as interleukin-1 (IL-1), IL-3, IL-6, and tumor necrosis factor-α (TNF- α) (1). These signals may influence the steroidogenic and mitogenic activities of adrenocortical cells *via* autocrine/paracrine processes (5). Indeed, ACTH has been shown to modulate cytokine production by adrenocortical cells, either positively (IL-6) or negatively (TNF-α) (45, 46). Intraadrenal cytokines may, thus, represent important effectors of ACTH capable of potentiating or attenuating its action on adrenocortical cells. However, there is currently no evidence that the impact of ACTH on adrenal cytokine synthesis may be involved in the pathogenesis of ACTH-dependent adrenal hyperplasia and/or hypercortisolism.

Several types of adrenocortical neoplasms are associated with illicit neuroendocrine differentiation of adrenocortical cells (47). This is especially true for APAs that have been shown to express synaptophysin, neuronal cell adhesion molecule, neuron specific enolase, and SV2 (48, 49). In addition, we have observed that APA cells may also abnormally synthesize 5-HT that is able to stimulate aldosterone secretion through activation of the overexpressed serotonergic type 4 (5-HT4) receptor (50, 51). Because APA cells also express high amounts of the ACTH receptor, i.e., the melanocortin receptor type 2 (MC2R) (51–53), it is conceivable that locally produced 5-HT may act as an amplifier of the stimulatory effect of ACTH on aldosterone secretion by APA tissues.

Inhibins and activin are dimeric peptides belonging to the TGF-β family. Inhibins are formed by combination of the α-subunit encoded by INHA and A or B isoform of the β-subunit, encoded by *INHBA* and *INHBB*, respectively. Alternatively, activin is a homodimer composed of two β-subunits. The action of activin is mediated by its specific receptors type I and II, and the intracellular proteins SMAD. Inhibins counteract the biological effects of activin by antagonizing activin type II receptor and formation of an inactive complex with the TGFβ type III receptor β-glycan. Adrenocortical cells are able to express both α and β subunits (54–56). In particular, the α-inhibin is expressed in the zona reticularis under the positive control of ACTH, whereas β-subunits are mainly present in the outer cortex. Both activin receptors and the inhibin co-receptor β-glycan are also detected in the adrenal cortex (55, 56). It has been demonstrated that ACTH stimulates secretion of inhibin A and B, without modifying production of activin A (55). These data indicate that ACTH also controls corticosteroidogenesis through modulation of the intraadrenal activins/inhibins ratio.

### INDIRECT EFFECTS OF ACTH ON CORTICOSTEROIDOGENESIS THROUGH NON-STEROIDOGENIC ADRENAL CELLS

The adrenal gland is surrounded by adipose tissue and its cortical region contains adipocytes either isolated or arranged in small islets (57). Like cells of the immune system, adipocytes release a wide panel of cytokines, suggesting that they could influence the adrenocortical function through a cell-to-cell communication process. For instance, adipocytes have been shown to activate aldosterone release by secreting soluble bioactive factors, which have not been yet characterized (57–59). Conversely, leptin exerts an inhibitory action on ACTH-induced corticosteroid secretion in human adrenocortical cells without affecting their viability and proliferation (60–63). It is possible that peri- and intraadrenal adipocytes may be controlled by ACTH and, thus, constitute a relay in the action of the hormone on the adrenal cortex. In support of this hypothesis, it has been reported that murine adipocyte cell lines and immortalized adipocytes express the MC2R, and ACTH regulate adipocyte functions in these models (64–66). In addition, patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, a condition which is associated with chronically high plasma ACTH levels, can present with adrenal myelolipoma (67–69), suggesting a role of ACTH in the development of lipomatous tissue inclusions in the adrenal glands. However, in contrast to the observations made in murine cells, human mature adipocytes only express low levels of the MC2R and ACTH does not influence lipolysis in the mature human adipose tissue (70).

### PARACRINE CONTROL OF ADRENAL STEROIDOGENESIS BY INTRAADRENAL ACTH

In the normal adrenal gland, chromaffin cells release detectable amounts of ACTH that is a major mediator in the corticomedullary functional interaction (71, 72) (**Figure 2**). This secretory process can be activated by corticotropin-releasing hormone (CRH) that is expressed in the adrenal medullary tissue (11). As chromaffin cells are also regulated by splanchnic nerves and proinflammatory cytokines, it seems possible that they may be important intermediates in the activation of the adrenal cortex during stress and inflammation. However, the physiological role of the paracrine control of corticosteroidogenesis by adrenomedullary ACTH remains unclear (3).

FIGURE 2 | Presence of ACTH in the normal human adrenal. ACTH immunoreactivity is exclusively located in some chromaffin cells (high panel), identified as chromogranin A (CGA) immunoreactive cells (low panel), in the medulla. ACTH was detected by using antibodies against the N-terminal region of the peptide. ZF, zona fasciculata; ZR, zona reticularis. Illustration of the data published in Ref. (9).

Conversely, clinicopathological studies have shown that paracrine interactions involving ACTH produced by chromaffin cells may play a role in the pathogenesis of hypercortisolism. Pheochromocytoma can occasionally produce ACTH resulting in ACTH-dependent Cushing's syndrome (73–75). Hypercortisolism seems to be mainly the consequence of the endocrine corticotropic action of ACTH whose plasma levels are typically elevated in this situation (76). Nevertheless, this mechanism may not be exclusive. In fact, hyperplasia of the adrenal cortex adjacent to pheochromocytoma has also been observed, indicating that ACTH originating from the pheochromocytoma tissue can stimulate adrenocortical cells in a paracrine manner (11, 77–80). Such a histological pattern is close to what is observed in corticomedullary mixed tumors that are composed of intermingled adrenocortical and pheochromocytoma tissues (81–84). These rare tumors are sometimes associated with hypercortisolism, suggesting that pheochromocytes release paracrine signals capable of activating glucocorticoid synthesis (81, 84). ACTH could be one of them although it is not excluded that catecholamines may exert a stimulatory action on cortisol production through illicit expression of adrenergic receptors in tumor adrenocortical cells (85, 86).

Alternatively, the occurrence of ectopic production of ACTH in the adrenal cortex has already been reported. Very rarely, ACTHpositive cells in the adrenocortical tissue can reveal adrenal micrometastases of an ACTH-secreting cancer. In the published cases, plasma ACTH levels were strongly elevated, as a result of ACTH secretion by the primary tumor (87–89). More surprisingly, a subpopulation of adrenocortical cells has been shown to produce detectable amounts of ACTH in various types of adrenal neoplasms. A first case of adrenocortical cortisol-secreting adenoma associated with production of ACTH by tumor cells, has been described in 2001 by Hiroi et al. (6). Illicit synthesis of ACTH was considered to result from abnormal pituitary differentiation of the tissue as witnessed by co-expression of 17-hydroxylase and pituitary homeobox factor-1 mRNAs by adrenocortical cells (6). At the ultrastructural level, tumor cells exhibited characteristics of both steroidogenic cells and neuroendocrine cells, and the tumor was, thus, referred to as an adrenocortical–pituitary hybrid adenoma. Bilateral macronodular adrenal hyperplasia (BMAH), a rare cause of primary adrenal hypercortisolism, has also been found to contain ACTH-producing cells. This observation was first reported by Pereira et al. who also noticed that ACTHpositive cells were labeled by antibodies to chromogranin A (CGA), suggesting that these cells may correspond to intracortical chromaffin cells (90). Subsequently, several teams reported expression of proopiomelanocortin (POMC) and ACTH in groups of adrenocortical cells in isolated BMAH cases (8, 10, 91) (**Figure 3**). In one case, it could be shown that ACTH-positive cells also expressed 17-hydroxylase but were negative for pituitary corticotroph markers (8). The role of ACTH in the pathogenesis of BMAH has been more extensively investigated in a large series of 30 cases (9). Adrenal hyperplasia samples expressed POMC mRNA at variable levels. Proconvertase 1, a protease implicated in the maturation of POMC into ACTH, was also visualized in clusters of adrenal cells, indicating that ACTH could be generated from POMC in the BMAH tissues. In agreement with this

the data published in Ref. (9).

finding, ACTH immunoreactivity was detected, as previously noticed, in some adrenocortical cells either isolated or arranged in small groups disseminated in the tissues. The presence of corticotropin can also be seen in some chromaffin cells of the adrenal medulla. Surprisingly, adrenocortical ACTH-positive cells also displayed characteristics of steroidogenic cells, such as the presence of numerous lipid inclusions and several markers of steroidogenic differentiation, including steroidogenic factor 1 (SF1), 17-hydroxylase, and the HDL-cholesterol receptor scavenger receptor B1 (SRB1). It could be, thus, concluded that they constitute a subcategory of adrenocortical steroidogenic cells characterized by an unusual capacity to synthesize ACTH. BMAH specimens were found to express very low levels of T-pit [a transduction factor which controls pituitary corticotrophs differentiation (92)], confirming that ectopic synthesis of ACTH in adrenocortical cells does not result from illicit corticotropic-like differentiation of the latter (9) but may rather be considered as an additional feature of neuroendocrine differentiation of the hyperplastic tissues (47, 93, 94). Interestingly, ACTH-containing cells were also positive for gonadal markers like the gonadal marker insulin-like 3 (INSL3), exhibiting thus a pseudo-gonadal phenotype (8, 9, 95, 96). This observation is concordant with expression of POMC and synthesis of ACTH previously reported in testicular Leydig cells and ovarian granulosa cells (97, 98). Because the adrenal glands and gonads both originate from a same tissue precursor, the adrenogonadal primordium, it is likely that the occurrence of these pseudo-gonadal cells in BMAH tissues may be the consequence of altered embryogenesis, explaining the bilaterality of the lesions.

Perifusion studies revealed that ACTH is released by BMAH tissues *in vitro* in a pulsatile mode (9), in agreement with older clinical studies showing a clear pulsatility of cortisol secretion in patients with BMAH (99). The ectopic secretion of ACTH by the hyperplastic adrenal glands has also been detected *in vivo* in two patients through adrenal vein catheterization (9). Taken collectively, these data suggested that, in BMAH tissues, intraadrenal ACTH may exert an autocrine/paracrine action to stimulate cortisol secretion, supplying therefore circulating ACTH that is suppressed by cortisol excess. This hypothesis was supported by statistical analyses showing positive correlations between ACTH and cortisol levels in BMAH culture medium. In addition, basal plasma cortisol concentrations measured *in vivo* were positively correlated with both the levels of POMC mRNA and the ACTH histological score in the tissues. Most importantly, MC2R antagonists, such as corticostatin and ACTH (7–38), were found to inhibit *in vitro* spontaneous and ACTH-evoked cortisol secretion by BMAH explants (9) (**Figure 3**). Clinical studies with MC2R antagonists are now mandatory to confirm that cortisol production is actually dependent on intraadrenal ACTH in patients with BMAH.

Bilateral macronodular adrenal hyperplasia tissues also constitute an interesting model for the study of the regulation of MC2R expression in the adrenal cortex. MC2R mRNA is globally underexpressed in BMAH samples versus normal adrenals (100). In fact, we could observe that, at variance with the normal adrenal cortex that is diffusely labeled by anti-MC2R antibodies, BMAH explants exhibit heterogeneous distribution of the receptor that appears highly expressed in the vicinity of clusters of ACTHproducing cells and more weakly at distance (**Figure 3**). Indeed, as previously established in the normal adrenal gland (101–103), MC2R seems to be upregulated by ACTH in BMAH tissues. In fact, MC2R mRNA levels were positively correlated with POMC mRNA rates and MC2R-like immunoreactivity was principally visualized in the vicinity of ACTH-positive cells (9). Interestingly, ACTH-producing cells were also found to express the receptor, suggesting that intraadrenal ACTH possibly exerts autocrine actions in BMAH.

All these data indicate that intraadrenal ACTH plays a pivotal role in the pathogenesis of hypercortisolism associated with BMAH. Deciphering the mode of regulation of ACTH production by BMAHs is, thus, essential for the comprehension of the pathophysiology of the disease. At variance with pituitary ACTH, intraadrenal ACTH does not seem to be regulated by cortisol, as suggested by the lack of action of dexamethasone and the glucocorticoid receptor antagonist RU486 on ACTH release by BMAH explants (9). Conversely, we noticed that ligands of various membrane receptors that are known to be abnormally expressed by BMAH cells, i.e., 5-HT, LH/hCG, and glucose-dependent insulinotropic peptide (GIP), are able to activate ACTH production from BMAH tissues *in vitro* (9). This surprising finding indicated that activation of illicit membrane receptors may stimulate cortisol production *via* two mechanisms, including a direct effect on corticosteroidogenesis, as previously shown in BMAH cell culture (93, 104), and an indirect action mediated by ACTH secretion (9). Consistently, MC2R antagonists were found to partially inhibit *in vitro* the cortisol response evoked by GIP in perifused BMAH samples. Taken together, these results suggest that intraadrenal ACTH may be regarded as a common intermediate and amplifier of the action of several illicit membrane receptors in BMAH tissues. Targeting the MC2R with specific antagonists may, thus, represent an efficient strategy for the treatment of BMAH-associated hypercortisolism. The pathophysiology links between intraadrenal ACTH and abnormally expressed receptors may be more complex and could form a complete auto-amplification loop in the hyperplastic tissues. In fact, although the presence of the LH and GIP receptors in adrenocortical cells may be simply considered as features of pseudo-gonadal differentiation of BMAH tissues, overexpression of some membrane receptors, such as 5-HT receptors, may result from local production from ACTH. This hypothesis is supported by intriguing observations recently performed in another type of adrenal hyperplasia associated with hypercortisolism, namely primary pigmented adrenocortical disease (PPNAD). The disease is caused in most patients by germline inactivating mutations that affect the *PRKAR1A* gene (105), resulting in constitutive activation of protein kinase A (PKA) in adrenocortical cells (106). Since MC2R are positively coupled to the cAMP/PKA pathway, it can be considered that *PRKAR1A* mutations partly mimic the action of ACTH on adrenal steroidogenic cells. Interestingly, PPNAD have been found to overexpress several types of 5-HT receptors, including the

FIGURE 4 | Putative pathophysiological mechanism responsible for bilateral macronodular adrenal hyperplasia. Both gonads and adrenals originate from the adrenogonadal primordium. It can be speculated that the causative genetic mutations may alter differentiation and/or separation of the adrenogonadal primordium leading to the presence of pseudo-gonadal cells in the adrenals. Secretion of ACTH by intraadrenal pseudo-gonadal cells may progressively stimulate, *via* activation of MC2R, both cortisol secretion and growth of the adrenocortical tissue, leading to bilateral adrenal hyperplasia associated with hypercortisolism. In parallel, sustained activation of the PKA pathway consecutive to activation of MC2R by intraadrenal ACTH may activate expression of some illegitimate receptors. Activation of steroidogenesis by ligands of illegitimate receptors further reinforces cortisol hypersecretion through both an intrinsic stimulatory action and an indirect effect *via* local release of ACTH.

5-HT4 receptor (107). It seems, thus, conceivable that abnormal expression of 5-HT receptors in BMAH tissues may result from exposure of adrenocortical cells to locally produced ACTH whose release is subsequently increased by the 5-HT signaling pathway.

There is now no doubt that BMAH is a genetically determined condition, *ARMC5* being a major susceptibility gene of the disease (108). It is now well established that the development of macronodular adrenal hyperplasia requires inactivation of the two *ARMC5* alleles, respectively, by the first germline mutation and a secondary somatic genetic event. Surprisingly, inactivation of *ARMC5* expression in the human adrenocortical cell line H295R, which reproduces the molecular defects observed in adrenocortical cells of patients with BMAH, results in a decrease in expression of steroidogenic enzymes (108, 109). It seems, thus, that a second line event is necessary for the emergence of hypercortisolism. We hypothesize that *ARMC5* mutations may alter differentiation and/or separation of the adrenogonadal primordium leading to the presence of pseudo-gonadal cells in the adrenal areas. Progressive expression of POMC and ACTH by these cells may then result in cortisol hypersecretion. Because illicit expression of ACTH has also been observed in non-ARMC5-mutated adrenal hyperplasias, it seems that abnormal differentiation of adrenocortical cells is a frequent histological feature in BMAH tissues whatever the causative firstline genetic defect (**Figure 4**).

### REFERENCES

### CONCLUSION

In addition to its well-known action on adrenocortical cells to promote steroidogenesis through activation of the MC2R, ACTH exerts multiple effects through the paracrine communication processes that occur in the adrenal gland in both physiological and pathophysiological conditions. ACTH can also be abnormally produced in adrenal neoplasms in which the hormone acts, thus, as an autocrine/paracrine factor to activate steroid secretion. This new pathophysiological concept opens novel avenues for the development of original pharmacological treatments of primary adrenal syndromes of steroid excess.