Regulation of human MC2-R gene expression by CREB, CREM, and ICER in the adrenocortical cell line Y1.
The MC2-Receptor (melanocortin 2 receptor, MC2-R) is a Gs-protein coupled receptor that is upregulated by its own ligand ACTH and by forskolin. The mechanisms regulating MC2-R expression are still unclear. We therefore investigated the role of the stimulatory transcription factors CREB and CREM and the inhibitory factor ICER for regulation of human MC2-R expression. We cotransfected mouse adrenocortical Y1 cells with luciferase reporter gene vectors containing full length and deleted human MC2-R promoter constructs with expression plasmids for CREB, CREBS133A, CREMtau, CREMtauS117A, or ICER. Direct protein-DNA interaction was investigated by EMSA. Wild type CREB did not significantly affect promoter activity due to high endogenous CREB activity. However, CREBS133A decreased forskolin stimulated MC2-R promoter activity by 48+/-5% (mean+/-SEM) while unstimulated values remained unchanged. CREMtau moderately increased basal and forskolin stimulated luciferase activity in a dose-dependent manner (maximum effect 252+/-24% and 186+/-13% VS. control vector, respectively). While this effect required the full length promoter, cAMP stimulation was retained in shorter constructs. ICER reduced basal luciferase activity in Y1 cells by 17+/-28%, but completely abolished forskolin stimulation. Although 5'-deletion constructs mapped the minimum promoter region required for ICER effect to the shortest -64/+40 construct, direct protein DNA interaction in this promoter region could not be identified by EMSA. Moreover, mutation of the SF-1 binding sites, which retained ICER dependent inhibition, excluded SF-1 to be required for this effect. We conclude from these data that transcription factors of the CREB/CREM/ATF family have a moderate effect on human MC2-R promoter activity, but seem to play a minor role in transmitting stimulation of the cAMP pathway to increased MC2-R expression.
Zwermann
O
O
Medizinische Klinik - Innenstadt, Ludwig-Maximilian-University, Munich, Germany. oliver.zwermann@med.uni-muenchen.de
Braun
A C
AC
Lalli
E
E
Sassone-Corsi
P
P
Beuschlein
F
F
Reincke
M
M
D000302:Adrenal Cortex / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D051786:Cyclic AMP Response Element Modulator / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic / Q000187:drug effects; D044103:Receptor, Melanocortin, Type 2 / Q000235:genetics*; D014162:Transfection
C115163:CREB1 protein, human; C497410:CREM protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D044103:Receptor, Melanocortin, Type 2; D051786:Cyclic AMP Response Element Modulator; D005576:Colforsin; D000242:Cyclic AMP