Functional analysis of rare polymorphisms within the Aryl Hydrocarbon receptor (AHR) gene in pituitary adenomas.

Abstract

The pituitary gland is integral in hormone secretion and regulation. Pituitary adenomas (PA) are the most frequent pituitary neoplasms, however molecular pathogenesis is largely unknown. The AHR is a ligand-activated transcription factor that regulates expression of various genes that mediate cellular response against xenobiotics, and is also involved in other physiological and pathological processes. Several AHR variants, particularly the Arg554Lys (rs2066853) and Val570Ile (rs4986826) have raised interest due to their location in ex on 10 of the AHR gene, which codes for the transactivation domain (TAD). The TAD domain plays a critical role in mediating transactivation activity of dioxin-responsive genes via recruitment of co-activators, hence suggesting that SNPs occurring within this region should interfere with AHR target gene expression. However, their exact functional role has not been established yet due to inconsistent results from different studies. Studies suggest that these mutations increase risk of developing P A, however functional analysis of these SNPs in a pituitary setting has never been carried out. In this research study, the two AHR variants were introduced in the wildtype AHR expression plasmid by site-directed mutagenesis (SDM). The wildtype and mutants AHR were introduced in GH3 cells by magnetofaction and were treated with 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). Functional analysis of transfected GH3 cells treated with TCDD was carried out using luciferase assays and real-time PCR to detect and quantify AHR-transcriptional activity. Cell proliferation of transfected and TCDD treated GH3 cells was measured using the MTT assay. In the absence and presence of low TCDD concentration, over-expression of AHR and AHR mutants did not affect the proliferative capacity of GH3 cells. Gene expression analysis and quantification analysis of AHR-target genes suggested that these AHR mutants might interfere with AHR target gene expression. However further studies are required to elucidate the precise mechanisms. Genotyping of the Arg554Lys in patients with PA gave a MAF of 3% vs 0% in neonatal controls using allele specific PCR.