Cyclic AMP-stimulated interaction between steroidogenic factor 1 and diacylglycerol kinase theta facilitates induction of CYP17.
In the human adrenal cortex, adrenocorticotropin (ACTH) activates CYP17 transcription by promoting the binding of the nuclear receptor steroidogenic factor 1 (SF1) (Ad4BP, NR5A1) to the promoter. We recently found that sphingosine is an antagonist for SF1 and inhibits cyclic AMP (cAMP)-dependent CYP17 gene transcription. The aim of the current study was to identify phospholipids that bind to SF1 and to characterize the mechanism by which ACTH/cAMP regulates the biosynthesis of this molecule(s). Using tandem mass spectrometry, we show that in H295R human adrenocortical cells, SF1 is bound to phosphatidic acid (PA). Activation of the ACTH/cAMP signal transduction cascade rapidly increases nuclear diacylglycerol kinase (DGK) activity and PA production. PA stimulates SF1-dependent transcription of CYP17 reporter plasmids, promotes coactivator recruitment, and induces the mRNA expression of CYP17 and several other steroidogenic genes. Inhibition of DGK activity attenuates the binding of SF1 to the CYP17 promoter, and silencing of DGK-theta expression inhibits cAMP-dependent CYP17 transcription. LXXLL motifs in DGK-theta mediate a direct interaction of SF1 with the kinase and may facilitate binding of PA to the receptor. We conclude that ACTH/cAMP stimulates PA production in the nucleus of H295R cells and that this increase in PA concentrations facilitates CYP17 induction.
School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230, USA.
Donghui
D
Li
Aarti N
AN
Urs
Jeremy
J
Allegood
Adam
A
Leon
Alfred H
AH
Merrill
Marion B
MB
Sewer
D000302:Adrenal Cortex / Q000166:cytology / Q000502:physiology; D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D019852:Diacylglycerol Kinase / Q000235:genetics / Q000378:metabolism*; D004790:Enzyme Induction; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism*; D054339:Steroidogenic Factor 1 / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D007527:Isoenzymes; C515057:NR5A1 protein, human; D010712:Phosphatidic Acids; D010743:Phospholipids; D054339:Steroidogenic Factor 1; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; D013254:Steroid 17-alpha-Hydroxylase; D019852:Diacylglycerol Kinase; D012319:RNA Polymerase II