Regulation of Human MC2-R Gene Expression by CREB, CREM, and ICER in the Adrenocortical Cell Line Y1

Abstract

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, CREMτ, CREMτS117A, 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. CREMτ 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.

Key words

adrenal - corticotropin - ACTH receptor - ACTH receptor promoter - corticotropin receptor expression

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