Endocrine Abstracts

Background: Germline mutations of the AIP tumour suppressor gene are associated with familial and sporadic pituitary adenomas, yet the tumorigenic mechanisms remain unclear. In addition, AIP protein expression in somatotroph adenomas from patients without AIP mutations correlates with clinical behaviour and somatostatin analogues responsiveness. Understanding the regulation of AIP gene expression will help uncover its pituitary tumour-suppressor role.

Aim: To identify AIP promoter sequences and study transcription factors (TFs) regulating AIP expression.

Methods: Bioinformatics and deletion constructs analysis of human AIP promoter-pGL4.16 luciferase reporters.

Results: A −323-5 deletion construct (relative to AIP start codon) had maximal unstimulated expression in GH3 cells. A longer construct, −1314-21 (ATG-relative) had significantly lower expression, suggesting the presence of silencer sequences between −1314 and −323. The previously reported AIP promoter mutation c.−270-269CG>AA (Igreja et al. 2010) had a significant inhibitory effect on luciferase expression from long (−1314-21) reporter constructs. Forskolin stimulation of GH3 cells transfected with WT or −270-269CG>AA mutant AIP-promoter reporter constructs showed a significant increase of activity for the long WT construct (−1314-21), that was not observed for the mutated variant. The short WT construct (−323-5) did not respond to forskolin and the mutation had no effect on basal and forskolin-stimulated activity. Bioinformatics identified numerous TF binding sites, including two distant CREs at positions −977 and −900, but none at −270-269.

Conclusions: We have mapped the maximal activity of the AIP promoter to a small proximal region, which includes a site that is mutated in a FIPA family. The effect of this mutation on cAMP activation of the promoter seems to be indirect and requires the presence of additional distal promoter sequence to the maximally active region.

Acknowledgements: S Radian was funded by Marie Curie (PIEF-GA-2011-303006) and IESP (European Society of Endocrinology) fellowships, with additional support from the TE227/2010 project (Romanian Ministry of Education).