The development and function of the endocrine system is highly dependent on second messengers in hormonal signaling. Advances in molecular and genetic studies and appreciation for the impact of second messengers on endocrine physiology and disease sparked important discoveries in signal pathway research especially over the past decades. The first second messenger to be identified was cyclic adenosine monophosphate (cAMP), described in 1958 by Earl Sutherland (1). The production and degradation of cAMP is regulated by adenyl cyclases (AC) and phosphodiesterases (PDEs), respectively (Figure 1) (1, 2). Recent studies have demonstrated a link between genetic alterations in PDEs and increased predisposition of tumor formation, particularly in the prostate, testis, and the adrenal cortex (3, 4). In primary bilateral macronodular adrenocortical hyperplasia (PBMAH), aberrant expression of several non-mutated G-protein-coupled receptors (GPCRs) showed that cAMP signaling could be increased without genetic mutations (5). In primary pigmented nodular adrenocortical disease (PPNAD), germline inactivating mutations of the protein kinase A regulatory subunit type 1 (PRKAR1A)-linked cAMP-dependent protein kinase (PKA) to adrenocortical tumors (ACTs) (6). Indeed, cAMP signaling dysregulation through expression defects or mutations appears to underlie the pathogenesis of most benign ACTs (5, 7).

Phosphodiesterases exist in over 100 isoforms and are derived from 21 genes separated into 11 PDE gene families (summarized in Table 1) (8–10). PDEs function through the hydrolyzation of cAMP (PDEs isoform 4, 7, and 8) and cyclic guanosine monophosphate (cGMP) (PDEs isoforms 5, 6, and 9) into AMP and GMP, respectively (8, 9). Dual-specificity PDEs (acting on both cAMP and cGMP with varying affinities) include the PDE1, PDE2, PDE3, PDE10, and PDE11 enzymes. All PDEs share a major structural feature; a conserved catalytic domain with about 300 amino acids located near the C-terminal regions, and a variable regulatory domain located in the N-terminal regions. PDEs vary in a number of ways, including a difference in substrate selectivity, tissue distribution, kinetic, and tissue expression (Table 1). The adrenal cortex expresses several isoforms of PDEs, including PDE2A that has been implicated in the downregulation of aldosterone production in adrenal zona glomerulosa cells and the regulation of the adrenocorticotropic hormone (ACTH)-induced increase in intracellular cAMP in the zona fasciculata cells (11).

Recent studies have demonstrated inactivating mutations and other germline variants in PDE11A and PDE8B in ACT causing Cushing syndrome (CS) (12–17). Here, we present a brief overview of the alterations of PDEs in ACTs. Given the breadth of this topic, we begin with a discussion of the cAMP-dependent signaling pathway in physiology, describe the current classification of ACTs, and then proceed with a discussion of PDE alterations in ACTs.

Briefly, GPCRs undergo conformational changes in response to various extracellular stimuli, such as ACTH (Figure 1). The first step in cAMP activation in adrenal cortex is the action of ACTH on its seven-transmembrane receptor, ACTHR [e.g., melanocortin 2 receptor (MC2R)]. This activation leads to the dissociation of the Gsα subunit (encoded by the GNAS gene) from the heterotrimeric G-proteins, activation of AC, generation of cAMP, and activating PKA (Figure 1). PKA exists as a tetrameric complex of two regulatory subunits (alpha and beta type 1 or alpha and beta type 2, encoded by PRKAR1A, PRKAR2A, PRKAR1B, and PRKAR2B) and two catalytic subunits (catalytic alpha and catalytic beta, encoded by PRKACA and PRKACB, respectively); the latter subunits are responsible for the phosphorylation of several enzymes and transcription factors downstream, including the cAMP-response element-binding protein (CREB). Abnormalities in some of these genes predispose to the formation of cortisol-producing ACTs (Figure 2) and increased steroid hormone secretion. The latter is often mediated by secondary factors regulated by PKA; in one study, silencing p54(nrb)/NONO expression in H295R human adrenocortical cells decreased the ability of the cells to increase intracellular cAMP production and subsequent cortisol biosynthesis in response to ACTH (18). The expression of multiple PDE isoforms, including PDE2A, PDE3A, PDE3B, PDE4A, PDE4D, and PDE11A, was induced in p54(nrb)/NONO knockdown cells, which suggests that these proteins may be responsible for the splicing and degradation of PDE transcripts (18). Studies in knockout mice (Table 2) have also pointed out to the significance of cAMP signaling-regulating genes in ACT formation.

In 1912, Cushing described the pituitary tumors that cause the condition that today bears his name (19). The causes of CS are broadly divided into ACTH-dependent and ACTH-independent disease. In 1984, the first description of Carney complex, a multiple neoplasia syndrome associated with spotty skin pigmentation, cardiac myxomas, pituitary tumors, and CS caused by PPNAD, provided the first insight into the genetic forms of ACT (20). In 1991, Weinstein et al. (21) described Gsα subunit (GNAS) mutations in individuals with McCune-Albright syndrome (MAS); MAS is classically associated with polyostotic fibrous dysplasia, café-au-lait skin spots, and precocious puberty but also with adrenal hyperplasia and/or tumors (21). MAS, albeit rare, appears to be the most frequent cause of CS among ACTH-independent adrenal hyperplasia in the infantile period (22). Kirschner et al. (23, 24) identified the regulatory subunit type 1A (R1α) of PKA (encoded by the PRKAR1A gene on chromosome 17q22-24) as the cause of PPNAD and CNC (25). PPNAD is the most frequent endocrine manifestation in CNC.

In 2006, the first association between PDE mutations and ACT, using a genome-wide association (GWA) study approach, was identified in patients with CS with PPNAD but without GNAS or PRKAR1A mutations (12). Three inactivating mutations in PDE11A were identified initially in patients, predominantly children, with micronodular adrenocortical hyperplasia (iMAD), a rare form of bilateral adrenocortical hyperplasia (BAH) leading to CS. Subsequent studies found other PDE mutations or functional variants, including alterations in PDE8B, in other ACTs, including PBMAH (14–17, 26, 27).

The new genetic findings influenced the diagnostic classification of these ACTs (5). In brief, three major ACTH-independent ACT subtypes exist: bilateral hyperplasias (BAH), adrenocortical adenomas (ACA), and adrenocortical cancer (ACC) (5). PBMAH, a form of BAH, is estimated to affect ~2% of patients with endogenous CS (5). This disease should be distinguished from secondary adrenocortical hyperplasia, which can occur after long-term stimulation by ACTH in Cushing disease (CS in the context of an ACTH-producing pituitary tumor) or ectopic ACTH secretion, predominantly from a neuroendocrine tumor (28). PBMAH is usually sporadic, but familial forms have been described (29). Cortisol-producing adenoma (CPA) is a benign subset of ACAs causing adrenal CS.

Bilateral adrenocortical hyperplasias can be broadly classified on the basis of the size of their nodules into micronodular (<1 cm in diameter) or macrocronodular (>1 cm in diameter) (5). The micronodular subtype is divided into pigmented (c-PPNAD, familial seen in CNC, or isolated, i-PPNAD) and not pigmented (e.g., iMAD) (5). PBMAH is the most common macronodular BAH [previously referred to as massive macronodular adrenocortical disease (MMAD) and ACTH-independent macronodular adrenocortical hyperplasia (AIMAH)] and is largely caused by mutations of the ARMC5 gene (30); several genes have been implicated in other forms of macronodular BAH, including GNAS, APC, and MEN1. Patients with MAS may develop a form of macronodular bilateral adrenal hyperplasia, called primary bimorphic adrenocortical disease (PBAD).

Mutations and variants in ACTs that lead to functional abnormalities of cAMP signaling have been reported in the GNAS, PRKAR1A, PDE11A, and PDE8B genes (9). Mutation-negative disease with activation of the cAMP pathway has been reported (31), suggesting that additional genetic (or perhaps epigenetic) “hits” may play a role in the pathogenesis of ACT.

Several genetic aberrations in the cAMP-dependent signaling pathway have been implicated in CPA. The most common genetic aberration in CPA is a somatic-activating mutations of PRKACA (c.617A>G/p.L206R) with an estimated incidence of ~42% (86 of 206 tumors studied to date) (50–52), with a predilection to younger patients with overt CS, suggesting a driver mutation role in tumorigenesis (44). Somatic mutations in GNAS were identified in 5–17% of CPA (53). The somatic allelic losses of PRKAR1A were described in 23% of CPA; these tumors were smaller in size and had a paradoxical increase in urinary cortisol levels after dexamethasone suppression (54), due to increased glucocorticoid receptor expression in ACT (55), as often observed in patients with c-PPNAD. Defects in Wnt-signaling have been reported in CPA, with CTNNB1 (p.S45P, p.S45F) in ~23% of cases (56). There are still many unknown genetic defects that lead to CPA formation.

There has been significant progress in PDE-related research over the past two decades. Genetic testing has uncovered several adrenocortical conditions that were linked to aberrations in PDE, often preceded by a long and insidious pre-diagnostic course. This has allowed earlier identification and better management of these lesions. However, there are several unanswered questions. PDE-related research is hampered by inherent (i.e., complexity of their structures, many intracellular interactions, and largely unknown function) and technical issues (e.g., there is lack of specific antibodies for the multiple isoform of each PDE). In addition to improving the characterization of PDE expression and function, future studies should also focus on the characterization of patients with various ACT phenotypes and PDE genotypes.

Preliminary results in using recombinant compounds to activate or inhibit the PDE11A structure may have important implications for drug development. Jäger et al. (47) produced approximately fourfold to fivefold increase in PDE11A-mediated hydrolysis of both cAMP and cGMP, with some degree of PDE11A specificity, with a cGMP analog (Rp-8-pCPTPET-cGMPS) bound to the PDE11A4 GAF domain (47). Furthermore, Ceyhan et al. (57) showed that BC11-28 and BC11-38 (potent and selective PDE11A inhibitors) in both yeast-based and enzyme assays had a >350-fold selectivity for inhibiting PDE11’s cGMP hydrolytic activity versus all other PDEs, while only BC11-38 inhibited PDE11A cAMP hydrolytic activity in H295R cells (57). Thus, a targeted molecular therapy approach for lesions related to defects in PDE may aid in the future management of affected or at risk patients.

Alterations in PDEs that lead to dysregulation of the cAMP-dependent signaling pathway have been linked to the development of ACT. These lesions are usually benign and represent an important group of genetic disorders causing CS. As genetic technology continues to revolutionize the field of endocrine genetics and as we continue to discover novel disease-causing genes on an unprecedented scale, new methods to rapidly assess the functional significance of PDE variants singly, or in combination, will evolve. In this review, we focused our discussion on the various genotypes and phenotypes of ACT due to alterations in PDE, particularly mutations in PDE8B and PDE11A. Although there has been significant progress in PDE-related research over the past two decades, there are as yet unidentified molecular causes for all of these lesions. We hope that one day targeted molecular therapies will replace adrenalectomy as the treatment of choice for these lesions.

All authors contributed equally to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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.