EXPERIMENTAL STUDY Clinical and molecular evidence for DAX-1 inhibition of steroidogenic factor-1-dependent ACTH receptor gene expression

Background: The ACTH receptor (ACTH-R) is a member of the seven transmembrane domain receptor super-family. In non-functional adrenal adenomas and adrenocortical carcinomas, ACTH-R expression is low. However, no inhibitory factor for ACTH-R expression has been defined to date. DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1) is a general repressor of steroid production, inhibiting steroidogenic factor-1 (SF-1)-dependent expression of multiple steroidogenic enzymes. The aim of this study was to inves-tigate whether ACTH-R gene transcription is affected by DAX-1 and whether this mechanism is involved in down-regulation of ACTH-R expression in adrenocortical tumors. Methods: We screened 22 adrenocortical tumors for ACTH-R and DAX-1 mRNA expression by North-ern blot. For in vitro analyses we co-transfected mouse Y1 adrenocortical carcinoma cells with the luciferase reporter gene vector pGL3 containing full-length constructs of human (h) or mouse (m) ACTH-R promoter together with a DAX-1 expression plasmid. These experiments were also performed using ACTH-R promoter 50-deletion constructs and constructs mutated at the SF-1-binding sites. Results: We found a negative correlation between DAX-1 and ACTH-R mRNA expression (R 20.47