Cyclic AMP-Stimulated Interaction between Steroidogenic Factor 1 and Diacylglycerol Kinase θ Facilitates Induction of CYP17

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-expression inhibits cAMP-dependent CYP17 transcription. LXXLL motifs in DGK-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.In the zonae fasciculata and reticularis of the human adrenal cortex, the biosynthesis of steroid hormones is activated by the peptide hormone adrenocorticotropin (ACTH), which activates a cyclic AMP (cAMP)-dependent signal transduction cascade, resulting in a rapid increase in hormone production and a sustained induction of the expression of steroidogenic genes. We have shown that cAMP-stimulated transcription of one of these steroidogenic genes, CYP17, is mediated by the binding of a complex containing steroidogenic factor 1 (SF1), p54 nrb , and polypyrimidine-tract-binding protein-associated splicing factor to the CYP17 promoter (93). The binding of this complex is concomitant with the cyclical association of several coregulatory proteins, including GCN5, steroid receptor coactivator 1, and ATPase-dependent chromatin remodeling factors (16).An integral regulator of CYP17 and most other steroidogenic genes in the adrenal cortex and gonads is SF1 (5, 94). SF1 is a nuclear receptor (NR) that plays a key role not only in steroidogenesis but also in endocrine development and sex differentiation (6,25,32,73,82,83). Targeted disruption of SF1 in mice resulted in adrenal and gonadal agenesis, absence of the ventromedial hypothalamic nucleus, and impaired expression of pituitary gonadotropins (42,69,118). The ability of SF1 to transactivate target genes has been shown to be regulated by phosphorylation (19,31,95,97), sumoylation (12, 54), acetylation (11,44,46), and protein-protein interactions (7,16,61,62,75,93,115,120).Recently we identified sphingosine (SPH) as an endogenous ligand for SF1 (109). SPH is bound to the receptor under basal conditions, and cAMP treatme...

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

“…In contrast, inhibition of DGK activity attenuates the binding of SF1-dependent gene expression, and silencing the expression of DGK expression inhibits cAMP-dependent CYP17A1 transcription. Finally, we have also shown that LXXLL motifs in DGK mediate a direct interaction of SF1 with the kinase and may facilitate ligand delivery ( 25 ). To date, studies have demonstrated that DGK is regulated by intracellular targeting ( 24 ), membrane lipids ( 12 ), protein-protein interactions ( 34 ), and intrinsic activity ( 12 ).…”

“…We have previously shown that cAMP signaling increases the transcription of CYP17A1 by stimulating the binding of SF1 to the CYP17A1 promoter ( 32,33 ). We have also shown that DGK regulates the production of PA, a ligand for SF1 that is produced in response to cAMP signaling ( 25 ). DGK acts as a coregulatory protein by binding to SF1 when the receptor is bound to chromatin.…”

“…Cells were sub-cultured onto 12-well plates and 24 h later treated with 0.4 mM Bt 2 cAMP for 1-24 h. Total RNA was extracted using Iso-RNA Lysis Reagent (5 Prime Inc., Gaithersburg, MD) and amplifi ed using a One-Step SYBR Green RT-PCR Kit (Thermo Fisher Scientifi c Inc., Waltham, MA) and the primer pairs described in Li et al ( 25 ). DGK expression was normalized to ␤ -actin content and calculated using delta-delta cycle threshold ( ⌬ ⌬ CT) method.…”

“…It has been postulated that the CRDs of the enzyme are required both for correct folding of the protein and for substrate presentation ( 23 ). Mutation of the CRD of DGK diminishes DAG-induced translocation of the enzyme to the plasma membrane ( 24 ); whereas the interaction between DGK and the nuclear receptor steroidogenic factor 1 (SF1) requires the PH domain ( 25 ). Diacylglycerol kinases (DGKs) are intracellular lipid kinases that phosphorylate diacylglycerol (DAG) to form phosphatidic acid (PA), which is linked to lipid metabolism and signaling (1)(2)(3).…”

Functional Proteomics: Mapping Lipid-Protein Interactomes

“…This is a compelling approach to decipher endogenous ligand occupancy of orphan nuclear receptor in an in vivo setting. Sewer and colleagues were able to identify several phospholipids bound to another orphan nuclear receptor, steroidogenic factor 1 (SF-1) (Li et al 2007) with roles in the regulation of steroidogenic hormones expression. SF-1 was immunoprecipitated from adrenocortical cells which express the receptor endogenously and phospholipids were analysed by LC-MS. Phosphatidic acid was found to be a major lipid bound to SF-1 and to activate the transcriptional activity of the receptor.…”