Exp Clin Endocrinol Diabetes 2005; 113 - V3_31
DOI: 10.1055/s-2005-862815

Regulation of ACTH-R gene expression by CREB, CREMt and ICER in the adrenocortical cell line Y1

O Zwermann 1, A Braun 2, E Lalli 3, F Beuschlein 2, M Reincke 1
  • 1Ludwig-Maximilian-Universität, Medizinische Klinik – Innenstadt, München
  • 2Albert-Ludwig-Universität, Medizin 2, Freiburg
  • 3University of Strasbourg, IGBMC, Illkirch, France

The ACTH-Receptor (ACTHr) is a Gs-protein coupled receptor that is up-regulated by it's own ligand ACTH and forskolin. The mechanisms regulating ACTHr expression following stimulation of the cAMP pathway are still unclear. We therefore investigated the role of the stimulatory transcription factors CREB and CREMt and the inhibitory factor ICER in regulation of ACTHr expression. We co-transfected mouse adrenocortical Y1 cells with luciferase reporter gene vectors containing full length and deleted ACTHr promoter with expression plasmids for CREB, CREBS133A, CREMt, CREMtS117A or ICER. CREBS133A and CREMtS117A lack the phosphorylation site required for stimulation of transcription. The three SF-1 binding sites in the ACTHrp were mutated by site directed mutagenesis, and deletion constructs with mutated binding sites were created. Direct protein-DNA interaction was investigated by EMSA. CREB WT and CREBS133A showed no effect on the ACTHr promoter constructs. CREMt increased moderately basal and forskolin stimulated luciferase activity dose dependently with a maximum at 252±24% (mean±SEM) and 186±13% compared to control vector. This effect could be competed by co-transfection with CREMS133A. ICER reduced basal and forskolin induced luciferase activity in Y1 cells by 17±28% and 68±4% respectively, and forskolin stimulation was completely abolished. As 5' deletion constructs mapped the site ICER action to the shortest construct EMSA was performed, excluding direct protein DNA interaction in this promoter region. Mutation of the SF-1 binding sites reduced promoter activity drastically, but inhibition of promoter activity by ICER was still visible. CREM had no effect in the mutated constructs. Northern Blots following transfection of the cells with CREM and ICER verified unchanged SF-1 levels. We conclude from these data:

  • 1. CREB is not involved in ACTHRp regulation following activation of the cAMP pathway.

  • 2. CREM moderately enhances ACTHRp activity without direct binding to the promoter.

  • 3. ICER is a strong SF-1 independent repressor of ACTHRp activity.