cAMP-specific PDE4 phosphodiesterases and AIP in the pathogenesis of pituitary tumors

Abstract

PDE4 cyclic nucleotide phosphodiesterases regulate cAMP abundance in cells and thereby regulate numerous processes, including cell growth and differentiation. The rat PDE4A5 isoform (human homologue PDE4A4) interacts with the AIP protein (also called XAP2 or ARA-9). Germline mutations in AIP occur in approximately 20% of patients with Familial Isolated Pituitary Adenoma (FIPA) and 20% of childhood-onset simplex somatotroph adenomas. We therefore examined the protein expression of PDE4A4 and the closely-related isoform PDE4A8 in normal human pituitary tissue and in pituitary adenomas. PDE4A4 had low expression in normal pituitary, but was significantly over-expressed in somatotroph, lactotroph, corticotroph and clinically non-functioning gonadotroph adenomas (P<0.0001 for all subtypes). Likewise, PDE4A8 was expressed in normal pituitary and was also significantly over-expressed in the adenoma subtypes (P<0.0001 for all). Among the different adenoma subtypes, corticotroph and lactotroph adenomas were the highest and lowest expressed for PDE4A4, respectively, whereas the opposite was observed for PDE4A8. Naturally occurring oncogenic variants in AIP were shown by a two-hybrid assay to disrupt the ability of AIP to interact with PDE4A5. A reverse-two-hybrid screen identified numerous additional variants in the TPR region of AIP that also disrupted its ability to interact with PDE4A5. The expression of PDE4A4 and PDE4A8 in normal pituitary, their increased expression in adenomatous pituitary cells where AIP is meant to participate, and the disruption of the PDE4A4-AIP interaction by AIP mutants may play a role in pituitary tumorigenesis.

Role of Phosphodiesterases on the Function of Aryl Hydrocarbon Receptor-Interacting Protein (AIP) in the Pituitary Gland and on the Evaluation of AIP Gene Variants

“…All the nonsense variants tested by this method so far (p.R81 * , p.Q164 * , p.Q217 * , and p.R304 * ) as well as one single amino acid deletion, p.Y248del, displayed null interaction with PDE4A5. Likewise, a significant reduction in the β-galactosidase activity was de-tected for the missense variants p.C238Y, p.R271W and p.V291M, and a borderline significant reduction was found for p.K241E, while the variants p.R16H, p.V49M, p.K103R, p.I257V, p.A299V, and p. R304Q did not have a significant effect [73]. In addition to impairing the interaction with PDE4A5, the experimentally created AIP variant p.R271A significantly reduced the ability to inhibit the enzymatic activity of PDE4A5, while other experimental missense variants tested (p.N236A and p.K266A) did not have an effect on PDE4A5 binding or enzymatic activity [76].…”

“…Enzymes of the PDE4 subfamily have a preferential cAMP hydrolytic activity (and very weak affinity for cGMP), are specifically inhibited by 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and, although ubiquitously expressed, the repertoire of isozymes varies in a tissue-specific manner [67,69,72]. PDE4A4 (encoded by the human PDE4A gene) is a 98.1 kDa protein expressed in the anterior pituitary in the somatotroph and gonadotroph cells (and poorly in lactotroph and corticotroph cells), while its expression is increased in somatotropinomas, prolactinomas, corticotropinomas, and FSH-positive NFPAs [23,73]. The rat homologue of this protein, PDE4A5, is well characterized, and contains a unique N-terminal region necessary for functional regulation (interactions with the LYN, FYN, and SRC tyrosine kinases) and membranous or perinuclear subcellular localization (the A-kinase anchoring protein 3 [AKAP3] binds this region), with 88 % identity with the human protein [74][75][76][77].…”

“…The AIP-PDE4A5 Interaction as a Functional Test for AIP Mutations Improved assessment of the pathogenicity of AIP mutations is paramount to provide adequate guidance for genetic counseling and clinical screening in AIP mutation carriers, which can lead to prospective diagnosis of pituitary adenomas [5,102]. Given that the AIP-PDE4A5 interaction and its functional consequences on the enzymatic function of PDE4A5 are well characterized, the evaluation of this interaction by a 2-H/β-galactosidase quantitative assay has proven to be a useful tool for testing AIP mutations detected in patients [32,73,76,103], complementing other in silico [21], in vitro [16,104], and in vivo [105] analyses.…”

“…Although usually concordant with other methods, the PDE4A5 binding assay allows a fine evaluation of a single protein function, which could increase its sensitivity, with the risk of introducing false positive results, given that PDE4A5 binding is not the only mechanism for the tumor suppressor function of AIP, and perhaps not even the most important one. For example, this method reported a loss of PDE4A5 binding for the AIP variant p.R304Q, which has been reported as nonpathogenic by other assays, complicating data interpretation [73]. While other testing methods are developed, our current recommendation is to rely on the combination of multiple methods for assessing the pathogenicity of AIP variants.…”

Novel Genetic Causes of Pituitary Adenomas

“…AIP has numerous interacting partners (12). It is co-chaperone to several heat shock proteins and nuclear receptors including the aryl hydrocarbon receptor, and it interacts with phosphodiesterase 4A4/5, which regulates the cAMP pathway (24)(25)(26); however, lack of AIP leads to reduced inhibitory G protein Gai-2 expression. The latter two aspects could be important to the tumorigenic role of AIP (27), as activation of the cAMP pathway is important in somatotroph tumorigenesis ( Fig.…”