Aip regulates cAMP signalling and GH secretion in GH3 cells

Abstract

Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been linked to predisposition to pituitary adenomas. However, the mechanism by which this occurs remains unknown. AIP interacts with a number of interesting proteins, including members of the cAMP signalling pathway that has been shown to be consistently altered in pituitary tumours. The functional role of Aip was investigated using both over-expression and knock down of Aip in GH3 cells. cAMP signalling and its downstream effectors, including GH secretion, were then investigated. cAMP signalling was analysed using cAMP assays, cAMP-response element-promoter luciferase reporter assays, real-time PCR and finally secreted GH quantification. Over-expression of wild-type (WT)-Aip reduced forskolin-induced cAMP signalling at the total cAMP level, luciferase reporter activity and target gene expression, when compared with empty vector and the non-functional R304X mutant. Additionally, GH secretion was reduced in WT-Aip over-expressing GH3 cells treated with forskolin. Knock down of endogenous Aip resulted in increased cAMP signalling but a decrease in GH secretion was also noted. Inhibition of phosphodiesterase activity using general and selective inhibitors did not completely ablate the effect of Aip on forskolin-augmented cAMP signalling. A mechanism by which Aip acts as a tumour suppressor, by maintaining a low cAMP signalling and concentration, is suggested. Mutations of Aip render the protein incapable of such activity. This effect appears not to be mediated by the AIP-PDE interaction, suggesting the involvement of other interacting partners in mediating this outcome.

AIP mutations impair AhR signaling in pituitary adenoma patients fibroblasts and in GH3 cells

“…However, both AIP mutants and Aip knockdown decreased GH secretion from kynurenine-treated GH3 cells. A similar observation has been previously reported after Aip knockdown in GH3 cells exposed to forskolin and IBMX (Formosa et al 2013). Thus, AIP haploinsufficiency, both after cAMP pathway and AhR pathway activation, decreases GH secretion from GH3 cells.…”

“…Secondly, activation of AhR signaling by kynurenine had antiproliferative action in this cell model. Finally, unlike cAMP pathway activation by forskolin (Formosa et al 2013), AhR activation by kynurenine did not modify basal GH secretion from untransfected GH3 cells expressing normal amounts of endogenous AIP (not shown). However, both AIP mutants and Aip knockdown decreased GH secretion from kynurenine-treated GH3 cells.…”

“…The molecular mechanisms of AIP-dependent pituitary tumorigenesis are elusive. Recent in vitro studies implicate the cAMP pathway, which seems to be activated by AIP deficiency (Formosa et al 2013, Tuominen et al 2014, but several lines of evidence also suggest the involvement of AhR. Most pathogenic AIP mutations alter the C-terminal region of the protein, particularly the last few amino acids required for AhR interaction (Bell et al 2000), resulting in loss of protein-protein interactions (Leontiou et al 2008, Morgan et al 2012.…”

Somatostatin analogues increase AIP expression in somatotropinomas, irrespective of Gsp mutations

“…A relationship between AIP, AHR and cAMP signalling, the best characterised pathway in GH-PA and an important target of SSA, is suggested by experimental evidence indicating that i) cAMP is a non-ligand activator of AHR able to induce transcriptional responses different from exogenous ligands (Oesch-Bartlomowicz et al 2005), ii) nucleocytoplasmic shuttling of AHR induced by cAMP is inhibited by PDE2, which is stabilised by AIP (de Oliveira et al 2007) and iii) AIP interactions with PDE4A5 can be disrupted by AIP mutations (Leontiou et al 2008). There is also very recent evidence that AIP reduces forskolininduced cAMP signalling in GH 3 cells, although forskolin did not influence AIP expression (Formosa et al 2013). However, intracellular cAMP concentrations have not been reported in the presence of AIP abnormalities in GH-PA and the potential effect of cAMP signalling on AIP expression in these tumours is unknown.…”

cAMP in the pituitary: an old messenger for multiple signals

“…Naturally occurring AIP-truncating mutations completely abolish the interaction with PDE4A5 (Bolger et al 2003, Leontiou et al 2008, Igreja et al 2010, Trivellin & Korbonits 2011. Another PDE isoform, PDE2A, expressed in human pituitary (Lennox et al 2011), has also been reported to bind to the C-terminal region of AIP and it has been shown that AIP has the opposite effect on PDE4A5 and PDE2A functions (de Oliveira et al 2007, de Oliveira & Smolenski 2009 Recently it has been shown that in GH3 cells AIP regulates cAMP signaling and GH secretion independently of the AIP-PDE interaction (Formosa et al 2013). The overexpression of the WT AIP, but not of a truncated mutant, reduces intracellular cAMP levels, the expression of cAMP target genes, and forskolin-induced GH secretion.…”