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 investigate 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 Northern 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 5'-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=-0.47, P<0.02). Accordingly, in vitro expression of DAX-1 significantly reduced hACTH-R and mACTH-R promoter activity by 89 and 55% respectively. DAX-1 inhibition was also present in the shortest construct of a series of 5'-deletion constructs of the human promoter extending from -64 to +40 bp relative to the transcription start site. Mutation of the SF-1-binding sites within the hACTH-R promoter resulted in reduced or abolished DAX-1 inhibition, arguing for a mechanism that involves SF-1 for DAX-1 inhibition.


CONCLUSIONS
These data support the concept that DAX-1 is a major repressor of ACTH-R gene expression in vitro and in vivo.
Zwermann
Oliver
O
Division of Endocrinology, Department of Internal Medicine II, University of Freiburg, Germany.
Beuschlein
Felix
F
Lalli
Enzo
E
Klink
Albrecht
A
Sassone-Corsi
Paolo
P
Reincke
Martin
M
D000236:Adenoma / Q000378:metabolism / Q000503:physiopathology*; D000310:Adrenal Gland Neoplasms / Q000378:metabolism / Q000503:physiopathology*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D018398:Homeodomain Proteins; D006801:Humans; D011401:Promoter Regions, Genetic / Q000502:physiology; D018043:Receptors, Corticotropin / Q000235:genetics*; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection
D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C539965:NR0B1 protein, human; C515057:NR5A1 protein, human; C539966:Nr0b1 protein, mouse; D018043:Receptors, Corticotropin; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D012097:Repressor Proteins; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; C515058:steroidogenic factor 1, mouse; D005576:Colforsin