Steroidogenic factor (SF1) is an orphan nuclear receptor that is essential for steroid hormone-biosynthesis and endocrine development. Recent studies have demonstrated that phospholipids are ligands for SF1. In the present study our aim was to identify endogenous ligands for SF1 and characterize their functional significance in mediating cAMP-dependent transcription of human CYP17. Using mass spectrometry we show that in H295R adrenocortical cells SF1 is bound to sphingosine (SPH) under basal conditions and that cAMP stimulation decreases the amount of SPH bound to the receptor. We also show that silencing both acid and neutral ceramidases using siRNA induces CYP17 mRNA expression, suggesting that SPH acts as an inhibitory ligand. In vitro analysis of ligand binding using scintillation proximity assays show that several sphingolipids and phospholipids, including phosphatidic acid (PA), can compete with [3H]SPH for binding to SF1, suggesting that SF1 may have more than one ligand and binding specificity may change with the changes in intracellular fluxes of phospholipids. Further, phosphatidic acid (PA) induces SF1-dependent transcription of CYP17 reporter constructs. Inhibition of diacyglycerol kinase (DAGK) activity using R59949 and silencing DAGK- expression attenuates SF1-dependent CYP17 transcriptional. We propose that PA is an activating ligand for SF1 and that cAMP-stimulated activation of SF1 takes place by displacement of SPH.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/7638 |
| Date | 22 November 2005 |
| Creators | Urs, Aarti N. |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Format | 824677 bytes, application/pdf |
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