ABSTRACT
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.
Subject(s)
Cyclic AMP/metabolism , Diacylglycerol Kinase/metabolism , Enzyme Induction , Isoenzymes/metabolism , Signal Transduction/physiology , Steroid 17-alpha-Hydroxylase/metabolism , Steroidogenic Factor 1/metabolism , Adrenal Cortex/cytology , Adrenal Cortex/physiology , Adrenocorticotropic Hormone/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Diacylglycerol Kinase/genetics , Gene Expression Regulation , Genes, Reporter , Humans , Isoenzymes/genetics , Phosphatidic Acids/metabolism , Phospholipids/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , Steroid 17-alpha-Hydroxylase/genetics , Steroidogenic Factor 1/genetics , Two-Hybrid System TechniquesABSTRACT
Transcription of the cytochrome P450 17 (CYP17) gene is regulated by cAMP-dependent binding of steroidogenic factor-1 (SF-1) to its promoter in the adrenal cortex. Using temporal chromatin immunoprecipitation and mammalian two-hybrid experiments, we establish the reciprocal presence of coactivators [general control nonderepressed (GCN5), cAMP response element-binding protein-binding protein, p300, p300/cAMP response element-binding protein-binding protein CBP associated factor, p160s, polypyrimidine tract associated splicing factor, and p54(nrb)], corepressors (class I histone deacetylases, receptor interacting protein, nuclear receptor corepressor, and Sin3A), and SWI/SNF (human homolog of yeast mating type switching/sucrose nonfermenting) and imitation SWI chromatin remodeling ATPases on the CYP17 promoter during transcription cycles in the H295R adrenocortical cell line. A ternary GCN5/SRC-1/SF-1 complex forms on the CYP17 promoter with cAMP-dependence within 30 min of cAMP stimulation, and corresponds with SWI/SNF chromatin remodeling. This complex is sensitive to the SF-1 antagonist sphingosine and results in decreased transcription of CYP17. GCN5 acetyltransferase activity and carboxy terminus binding proteins alternatively mediate disassembly of the complex. This work establishes the temporal order of cAMP-induced events on the promoter of a key steroidogenic gene during SF-1-mediated transcription.
Subject(s)
Cell Cycle Proteins/metabolism , Cyclic AMP/metabolism , Histone Acetyltransferases/metabolism , Homeodomain Proteins/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Octamer Transcription Factors/metabolism , RNA-Binding Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Sphingosine/metabolism , Steroid 17-alpha-Hydroxylase/metabolism , Transcription Factors/metabolism , Acetylation , Amino Acid Sequence , Bucladesine/pharmacology , Cell Line , Chromatin Assembly and Disassembly , DNA Polymerase II/metabolism , DNA-Binding Proteins , Humans , Molecular Sequence Data , Nuclear Receptor Coactivator 1 , PTB-Associated Splicing Factor , Promoter Regions, Genetic , Protein Binding , Signal Transduction , Steroid 17-alpha-Hydroxylase/genetics , Steroidogenic Factor 1 , Transcriptional Activation , p300-CBP Transcription FactorsABSTRACT
In the human adrenal cortex, ACTH activates steroid hormone biosynthesis by acutely increasing cholesterol delivery to the mitochondrion and chronically increasing the transcription of steroidogenic genes (including CYP17) via a cAMP-dependent pathway. In the present study, we characterized the role of sphingolipids in ACTH-dependent steroidogenesis. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) and the content of several sphingolipid species quantified by mass spectrometry. Both ACTH and Bt2cAMP decreased cellular amounts of several sphingolipids, including sphingomyelin, ceramides, and sphingosine and stimulating the activity of sphingosine kinase and increasing the release of sphingosine-1-phosphate (S1P) into the media. S1P increased CYP17 mRNA expression by promoting the cleavage and nuclear localization of sterol regulatory element binding protein (SREBP) 1. Chromatin immunoprecipitation assays revealed that Bt2cAMP and S1P increased acetylation of histone H3 and promoted binding of SREBP1 to the -520/-331 region of the CYP17 promoter. In summary, our studies demonstrate a role for sphingolipid metabolism and SREBP1 in ACTH-dependent CYP17 regulation and steroidogenesis.
