cAMP-stimulated transcription of DGK θ requires steroidogenic factor 1 and sterol regulatory element binding protein

Abstract

Diacylglycerol kinase (DGK) is a lipid kinase that phosphorylates diacylglycerol to form phosphatidic acid (PA). We have previously shown that PA is a ligand for the nuclear receptor steroidogenic factor 1 (SF1) and that cAMPstimulated expression of SF1 target genes requires DGK . In this study, we sought to investigate the role of cAMP signaling in regulating DGK gene expression. Real time RTPCR and Western blot analysis revealed that dibutyryl cAMP (Bt 2 cAMP) increased the mRNA and protein expression, respectively, of DGK in H295R human adrenocortical cells. SF1 and sterol regulatory element binding protein 1 (SREBP1) increased the transcriptional activity of a reporter plasmid containing 1.5 kb of the DGK promoter fused to the luciferase gene. Mutation of putative cAMP responsive sequences abolished SF1- and SREBP-dependent DGK reporter gene activation. Consistent with this finding, chromatin immunoprecipitation assay demonstrated that Bt 2 cAMP signaling increased the recruitment of SF1 and SREBP1 to the DGK promoter. Coimmunoprecipitation assay revealed that SF1 and SREBP1 interact, suggesting that the two transcription factors form a complex on the DGK promoter . Finally, silencing SF1 and SREBP1 abolished cAMP-stimulated DGK expression. Taken together, we demonstrate that SF1 and SREBP1 activate DGK transcription in a cAMPdependent manner in human adrenocortical cells. Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.