Background: AIP mutations cause sporadic and familial pituitary adenomas, but establishing the pathogenic role of missense AIP variants with unknown significance is difficult. The AIP interaction partner AHR a xenobiotic-activated transcription factor regulates transcription of xenobiotic-metabolising enzymes, mediates xenobiotic toxicity, and has been implicated in tumorigenesis.
Aim: To describe the effect of AIP dosage and mutations on AHR-dependent transcription and use it to assess AIP variant pathogenicity.
Materials and methods: Aip was knocked-down (KD) by Aip-siRNA transfection or Aip-shRNA lentiviral transduction in the GH3 rat somatotrophinoma cell line. WT or mutant human-AIP were over-expressed by transfection. AIP protein levels were assessed by immunoblotting. Expression levels of Aip, Ahr, and Cyp1a1 (an AHR target gene) were measured by RT-qPCR. AHR-dependent transcription was stimulated with an endogenous ligand, 6-formylindolo[3,2-b]carbazole (FICZ).
Results: Immunoblotting confirmed endogenous Aip KD and efficient expression of transfected human-AIP unaffected by RNA-interference. siRNA Aip-KD caused a significant reduction of Ahr and Cyp1a1 mRNA levels, in both FICZ and vehicle-treated cells. In stable Aip-KD GH3 cells, reduced Cyp1a1 expression was rescued by WT-AIP transfection, but not by the pathogenic p.R304* truncation mutant. The p.C238Y and p.R271W pathogenic missense AIP variants did not rescue Cyp1a1 expression, while the likely pathogenic (based on clinical data) p.R304Q and non-pathogenic p.R16H displayed an intermediate rescue.
Conclusions: AIP deficiency through KD or mutation is associated with reduced AHR expression and transcriptional activity. The effect of AIP variants on AHR-dependent transcription is a potential measure of mutation pathogenicity.
Acknowledgments: MdOA was 20142015 Science without Borders programme (SwB-CNPq) student; D S Soare has received a Society for Endocrinology 2014 Summer studentship; and S Radian was Marie Curie postdoctoral fellow (REGAIP-303006).